Compositions with hematopoietic and immune activity

ABSTRACT

The present invention provides methods of using Bv8 and EG-VEGF polypeptides and nucleic acids to promote hemtopoiesis. Also provided herein are methods of screening for modulators of Bv8 and EG-VEGF activity. Furthermore, methods of treatment using Bv8 and EG-VEGF polypeptides or Bv8 and EG-VEGF antagonists are provided.

GENENTECH, INC. et al., a United States national and resident, is filingthis application as a PCT application, claiming prior to U.S.Provisional Application Nos. 60/454,462 filed 12 Mar. 2003 and60/511,390 filed 14 Oct. 2003.

BACKGROUND OF THE INVENTION

Bv8 is a small protein that was originally isolated from the skinsecretions of the frog Bombina variegata (Mollay et al., Eur. J.Pharmacol., 374:189-196 (1999)). Bv8 belongs to a structurally relatedclass of peptides that includes endocrine gland derived vascularendothelial growth factor (EG-VEGF) (LeCouter et al., Nature,412:877-884 (2001)). A distinguishing structural motif of thesemolecules is a colipase-fold, where 10 cysteine residues form fivedisulfide bridges within a conserved span.

Bv8 and EG-VEGF are homologs of vascular endothelial growth factor(VEGF), an angiogenic factor known to have an important role in tumorgrowth and survival. Both Bv8 and EG-VEGF have been identified asangiogenic factors with selective activities for endothelial cells ofspecific tissues. EG-VEGF promoted proliferation, migration, survival,and fenestration in cultured adrenal capillary endothelial cells andinduced angiogenesis in ovary and testis. LeCouter et al., 2001, Nature,412:877-884; LeCouter et al., 2003, Proc. Natl. Acad. Sci. USA,100:2685-2690.

Like EG-VEGF, Bv8 promoted proliferation, survival, and migration ofadrenal cortical capillary endothelial cells and induced angiogenesis intestis. LeCouter et al., 2003, Proc. Natl. Acad. Sci. USA,100:2685-2690. The testis exhibits relatively high turnover ofendothelial cells. Thus, Bv8 and EG-VEGF, along with other factors suchas VEGF, are considered to be important in maintaining the integrity andregulating proliferation of the blood vessels in the testis.

VEGF is an angiogenic factor known to have an important role in tumorgrowth and survival. Because Bv8 and EG-VEGF are angiogenic factors withselective activity for specific tissues, it is desirable to furthercharacterize the molecules.

SUMMARY OF TIE INVENTION

The present invention is based on the identification of novel expressionand activities of Bv8 and EG-VEGF in hematopoietic stem cells (HSCs),lineage-committed blood progenitor cells, and lymphocytes. Inparticular, as described in detail herein, Bv8, EG-VEGF, and theirreceptors are expressed in bone marrow HSCs, peripheral blood leukocytes(PBLs), as well as many hematological malignant cell lines. Both invitro and in vivo experiments showed that Bv8 and EG-VEGF are capable ofpromoting colony formation of bone marrow mononuclear cells andspleen-derived progenitor cells, increasing populations of white bloodcells, and promoting activation of B lymphocytes and T lymphocytes.Accordingly, Bv8 nucleic acids and polypeptides, EG-VEGF nucleic acidsand polypeptides, or combinations thereof can be used in a number ofassays and in diagnosis and treatment of conditions associated withhematopoiesis, neutropenias, immunodeficiency disorders, and autoimmunedisorders.

Receptors for BV8 and EG-VEGF have now been found on bone marrowhematopoietic stem cells (CD34+) and on lineage committed progenitorcells (CD34+). BV8 and/or EG-VEGF induce proliferation of CD34+ myeloidand lymphoid progenitor cells. This proliferation leads to an increasein the number of white blood cells, including B cells, T cells, and inparticular, neutrophils. BV8, EG-VEGF, and their agonists aretherapeutically useful, for example, in the treatment ofimmunodeficiency disorders, such as lymphopenia, neutropenia, andothers. These molecules are also useful to promote hematopoieticrecovery after myelosupression, for example as induced by chemotherapy.

Various leukemic cells, such as ALL, AML, MPD, CML, and MDS cells, havenow also been found to express receptors for BV8 and EF-VEGF.Antagonists of the growth factors EG-VEGF and/or BV8 are useful toinhibit proliferation of these leukemic cells.

BV8 and/or EG-VEGF have also surprisingly been found to induce B and Tactivation. Agonists and antagonists of these molecules aretherapeutically useful to modulate an immune response. BV8, EG-VEGF, andtheir agonists are useful to and induce proliferation and activation ofT cells in immunocompromised individuals, for example, in HIV patients.Antagonists of these molecules are therapeutically useful to inhibit animmune response, for example, those associated with an autoimmunedisorder.

Proliferation of Bone Marrow Cells

In one aspect, the present invention provides a method of inducing bonemarrow cell proliferation. In one embodiment, the method comprisescontacting BM cells with Bv8, EG-VEGF, or a combination thereof in anamount effective to induce proliferation of the cells. In anotherembodiment, the method comprises introducing a polynucleotide sequenceencoding Bv8, EG-VEGF, or a combination thereof, into BM cells in anamount effective to induce BM cell proliferation.

In one embodiment, the Bv8 and/or EG-VEGF is a native sequencepolypeptide. Preferably, the native sequence Bv8 polypeptide is a nativehuman Bv8 polypeptide. The native human Bv8 polypeptide may comprise theamino acid sequence of SEQ ID NO: 2 or of SEQ ID NO: 4. In anotherembodiment, the native sequence Bv8 polypeptide comprises the amino acidsequence of SEQ ID NO: 6. In another embodiment, the Bv8 polypeptide iscapable of binding heparin. Preferably, the native sequence EG-VEGFpolypeptide is a native human EG-VEGF polypeptide. The native humanEG-VEGF polypeptide may comprise the amino acid sequence of SEQ ID NO:8.In another embodiment, the native sequence EG-VEGF comprises the aminoacid sequence of SEQ ID NO: 10. In yet another embodiment, the Bv8and/or EG-VEGF is an immunoadhesin. In a further embodiment, the Bv8and/or EG-VEGF is chimeric.

In one embodiment, Bv8/EG-VEGF promote proliferation of BM hematopoieticstem cells and/or lineage committed progenitor cells. The lineagecommitted progenitor cells are generally of the myeloid and/or lymphoidlineage.

In a further aspect, the invention provides a method of maintainingspecific blood cell populations in a patient in need of such cells. Inone embodiment this method comprises contacting the cells with Bv8,EG-VEGF, or a combination thereof, in an amount effective to promoteproliferation of said cells, thereby maintaining the population thereofIn another embodiment this method comprises introducing a polynucleotidesequence encoding Bv8, EG-VEGF, or a combination thereof, into cells inan amount effective to enhance cell survival. This method may furthercomprise introducing a polynucleotide sequence encoding VEGF to thecells. In one aspect, the particular cell types in need are leukocytes,preferably neutrophils, B lymphocytes, CD4+ T lymphocytes, and/or CD8+ Tlymphocytes. In another aspect, the patients suffer from neutropenia,lymphopenia, or an immunodeficiency disorder, and are therefore in needof neutrophils, B lymphocytes, CD4+ T lymphocytes, and/or CD8+ Tlymphocytes.

Treatment of Abnormal Hematopoiesis

In a further aspect, the present invention provides a method of treatinga mammal for a condition associated with abnormal hematopoiesis. In oneembodiment, the method preferably comprises administering to the mammala composition comprising Bv8, EG-VEGF, or a combination thereof, or anagonist or antagonist thereof, in an amount effective to treat thecondition. The mammal is preferably human.

In one aspect, the composition comprising Bv8, EG-VEGF, or a combinationthereof, used in any of the methods of the invention comprises a nativesequence Bv8 polypeptide and/or native sequence EG-VEGF polypeptide.Preferably, the native sequence Bv8 polypeptide is a native human Bv8polypeptide. The native human Bv8 polypeptide may comprise the aminoacid sequence of SEQ ID NO: 2 or the amino acid sequence of SEQ ID NO:4. In another embodiment, the native sequence Bv8 comprises the aminoacid sequence of SEQ ID NO: 6. In another embodiment, the Bv8polypeptide binds heparin. Preferably, the native sequence EG-VEGFpolypeptide is a native human EG-VEGF polypeptide. The native humanEG-VEGF polypeptide may comprise the amino acid sequence of SEQ ID NO:8.In another embodiment, the native sequence EG-VEGF comprises the aminoacid sequence of SEQ ID NO:10.

Treatment of Hematological Disorders

In yet a further aspect the invention provides a method of treating ahematological disorder in a mammal, preferably a human. In oneembodiment the method comprises administering to the mammal a Bv8antagonist, EG-VEGF antagonist, or a combination thereof, in an amounteffective to inhibit cell proliferation. In one embodiment, thehematological disorders treatable with the methods of the inventioninclude various leukemia, myeloproliferative disorders, myelodysplasticdisorders, lymphoproliferative disorders, and lymphodysplasticdisorders. Preferably, the hematological disorders is selected from thegroup consisting of acute myeloid leukemia (AML), chronic myelogenousleukemia (CML), acute lymphoblastic leukemia (ALL), multiple myeloma,T-cell lymphoma, polycythaemia vera (PV), essential thrombocythaemia(ET), and myeloid metaplasia (myelofibrosis), and the like.

Treatment of Immune Disorders

A further aspect of the invention provides a method for treating animmunodeficiency disorder in a mammal, preferably human, byadministering Bv8, EG-VEGF, or a combination thereof Patients sufferingan immunodeficiency disorder lack B and T lymphocytes and are in need ofenhanced B lymphocyte and/or T lymphocyte populations. Bv8, EG-VEGF, ora combination thereof can be provided to increase B and T cellpopulation. The immunodeficiency disorder may be primary or secondary.In one embodiment, the secondary immunodeficiency disorder is acondition associated with an infectious disease including humanimmunodeficiency virus (HIV) or hepatitis. In another embodiment, theimmunodeficiency disorder is a condition associated with theadministration of an immunosuppressive agent, such as a therapeuticagent including but not limited to, 5 fluorouracil, vincristine,cisplatin, oxoplatin, methotrexate, 3′-azido-3′-deoxythymidine,paclitaxel, doxetaxel, an anthracycline antibiotic, or mixtures thereofhaving a secondary immunosuppressive effect.

A further aspect of the invention provides a method for treating anautoimmune disorder in a mammal, preferably human. Bv8 antagonists,EG-VEGF antagonists, or combinations thereof, can be useful to treatautoimmune disorders where a decrease in the number of activated Bcells, CD4+ T cells, and/or CD8+ T cells is desirable. Specificembodiments include using the agents and compositions provided herein totreat type II, III, and IV hypersensitivity responses associated withautoimmune disorders. In one embodiment, the method comprisesadministering to a mammal, a Bv8 antagonist, EG-VEGF antagonist, or acombination thereof, in an amount effective to inhibit the autoimmunedisorder. In another embodiment, the method comprises administering aBv8 antagonist, EG-VEGF antagonist, or a combination thereof, to apatient in an amount effective to inhibit proliferation of CD4+lymphocytes and/or CD8+ T lymphocytes.

Modulation of Immune Response

A further aspect of the invention provides a method for modulating animmune response. Bv8, EG-VEGF, or a combination thereof, or an agonistthereof, can be administered to activate B lymphocytes, CD4+ Tlymphocytes, and/or CD8+ T lymphocytes. In one embodiment, Bv8, EG-VEGF,or a combination thereof, or an agonist thereof, can be administered toselectively promote or inhibit the proliferation of CD4+ T lymphocytesand/or CD8+ T lymphocytes.

Bv8 and EG-VEGF induce cytokine production in CD4+ T lymphocytes andCD8+ T lymphocytes. In one embodiment, a Bv8, EG-VEGF, an agonistthereof, or a combination thereof, that induces IL-2 synthesis in CD4+ Tlymphocytes can be used to induce the proliferation of CD4+ lymphocytes.In another embodiment, Bv8, EG-VEGF, an agonist thereof, or acombination thereof, that induces IFN-γ in CD4+ T lymphocytes can beadministered to inhibit the proliferation of CD4+ T lymphocytes.

Antagonists

Bv8 antagonists and EG-VEGF antagonists useful in the invention can beany composition capable of blocking, interfering, or minimizing Bv8and/or EG-VEGF activities. These antagonists include anti-Bv8 and/oranti-EG-VEGF antibodies or fragments thereof, truncated peptides capableof binding to Bv8 and/or EG-VEGF receptors without eliciting signaltransduction activities, soluble Bv8 and/or EG-VEGF receptors capable ofsequestering Bv8 and/or EG-VEGF peptides, anti-Bv8 and/or anti-EG-VEGFreceptor antibodies or small molecules capable of interfering with Bv8or EG-VEGF receptor activities. In one embodiment, the Bv8 or EG-VEGFreceptor is Bv8/EG-VEGF Receptor-1 and/or Bv8/EG-VEGF Receptor-2. BothBv8 and EG-VEGF bind to Receptor 1 and Receptor 2 as described morefully in the detailed description.

Article of Manufacture

In a still further aspect, the invention provides an article ofmanufacture comprising a container, Bv8 and/or EG-VEGF, and instructionsfor using the Bv8 and/or EG-VEGF. In one embodiment, the instructionsare for using the Bv8 and/or EG-VEGF to treat a condition that isassociated with abnormal hematopoiesis. In another embodiment, theinstructions are for using the Bv8 and/or EG-VEGF to treat a conditionthat is associated with immunodeficiency disorders. In another aspectthe invention provides an article of manufacture comprising a container,a Bv8 antagonist and/or EG-VEGF antagonist, and instructions for usingthe Bv8 antagonist and/or EG-VEGF antagonist. In one embodiment theinstructions are for using the Bv8 antagonist and/or EG-VEGF antagonistto treat hematological disorders. In another embodiment, theinstructions are for using the Bv8 antagonist and/or EG-VEGF antagonistto treat immunodeficiency disorders. In another embodiment, theinstructions are for using the Bv8 antagonist and/or EG-VEGF antagonistto treat autoimmune disorders.

Method to Identify Antagonist

Another aspect of the invention provides a method for identifying a Bv8or EG-VEGF antagonist by contacting a candidate compound with Bv8 orEG-VEGF, determining the effect of the compound on a Bv8 or EG-VEGFbiological activity, and identifying an antagonist where a Bv8 orEG-VEGF biological activity is inhibited. In one embodiment, a Bv8antagonist is identified by its inhibition of the ability of Bv8 tostimulate endothelial cell proliferation. In another embodiment, a Bv8or EG-VEGF antagonist is identified by its inhibition of the ability ofBv8 or EG-VEGF to promote endothelial cell survival.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the nucleotide sequence (SEQ ID NO: 1) of a cDNA encoding ahuman Bv8 homologue. Positions of the respective start codon (“atg”beginning at nucleic acid position 11) and stop codon (“taa” beginningat nucleic acid position 398) are presented in bold font and underlined.

FIG. 2 shows the amino acid sequence (SEQ ID NO: 2) of a human Bv8homologue polypeptide as derived from the coding sequence of SEQ IDNO: 1. A putative signal sequence comprises amino acids 1 through 21.

FIG. 3 shows the nucleotide sequence (SEQ ID NO: 3) of a cDNA encodingan alternatively spliced version of the human Bv8 homologue. Positionsof the respective start codon (“atg” beginning at nucleic acid position11) and stop codon (“taa” beginning at nucleic acid position 394) arepresented in bold font and underlined.

FIG. 4 shows the amino acid sequence (SEQ ID NO: 4) of a human Bv8homologue polypeptide derived from the coding sequence of SEQ ID NO: 3.

FIG. 5 shows the polynucleotide sequence (SEQ ID NO: 5) of a mouse Bv8homologue. Positions of the respective start codon (“atg” beginning atnucleic acid position 46) and stop codon (“tag” beginning at nucleicacid position 382) are presented in bold font and underlined.

FIG. 6 shows the amino acid sequence (SEQ ID NO: 6) of a mouse Bv8homologue polypeptide derived from the coding sequence of SEQ ID NO: 5.

FIG. 7 shows an alignment of mouse (SEQ ID NO: 41) and human Bv8homologues (SEQ ID NO: 2). A potential heparin-binding domain is boxed.This domain is not present in an alternatively spliced transcript. Aputative signal sequence is underlined. The mouse and human Bv8homologues are approximately 96% identical.

FIG. 8 shows the polynucleotide sequence (SEQ ID NO:7) of a cDNAencoding human native sequence EG-VEGF.

FIG. 9 shows the amino acid sequence (SEQ ID NO:8) of a human nativesequence EG-VEGF polypeptide derived from the coding sequence of SEQ IDNO:7.

FIG. 10 shows the polynucleotide sequence (SEQ ID NO:9) of a cDNAencoding a native mouse EG-VEGF polypeptide (SEQ ID NO:10).

FIG. 11 shows an alignment of human native EG-VEGF polypeptide (SEQ IDNO:8) and native mouse EG-VEGF polypeptide (SEQ ID NO:8).

FIG. 12 shows an alignment of the amino acid sequences of human Bv8homologue (amino acids 28-108 of SEQ ID NO:4) and human EG-VEGF (aminoacids 20-105 of SEQ ID NO:10). The signal sequence is not shown foreither molecule. Human Bv8 is approximately 60% identical to humanEG-VEGF.

FIG. 13 shows the results of a dot blot hybridization of RNA assay thatreveals a hBv8 signal in bone marrow, PBLs, as well as testis.

FIGS. 14A-D are photographs showing results of in situ hybridizationstudies that reveal restricted expression of Bv8 in neutrophils andassociated infiltrating cells. FIGS. 14A and B show Bv8 expression intonsillitis, where FIG. 14A shows hematoxylin-eosin staining of thetissue and FIG. 14B shows Bv8 expression in the same tissue sample using³³P-labeled probes. FIGS. 14C and D show Bv8 expression in appendicitis,where FIG. 14C shows a hematoxylin-eosin staining of the tissue and FIG.14D shows Bv8 expression in the same tissue sample using ³³P-labeledprobes.

FIGS. 15A-D are graphs showing the results of real time quantitative PCRexpression analysis of Bv8 and its receptors in various tissues andcells. FIG. 15A shows that Bv8 is strongly expressed in bone marrow aswell as testis; FIG. 15B shows differential expression of Bv8 in varioustypes of hematopoietic cells; and FIGS. 15C and D show hematopoieticcell expression of Bv8/EG-VEGF receptor-1 (FIG. 15C) and Bv8/EG-VEGFreceptor-2 (FIG. 15D).

FIG. 16 is a bar graph showing the results of real time quantitative PCRexpression analysis of Bv8 and Bv8/EG-VEGF receptor-1 and Bv8/EG-VEGFreceptor-2 in various leukemia cell lines (A) HL60 CML; (B) K562CML; (C)Hel-92 erythroleukemia; (D) TF-1 pancytopenia; (E) KG-1 AML.

FIGS. 17A-B are graphs showing colony formations in bone marrowmononuclear cultures in vitro, in the presence of various growthfactors. FIG. 17A shows that Bv8 (at both 5 nM and 50 mM) increasescolony formation in mouse bone marrow mononuclear cells. FIG. 17B showsthat Bv8, similar to EG-VEGF, increases colony formation of certaintypes of myeloid progenitor cells in human bone marrow mononuclear cellcultures. Ct refers to complete medium as described in Example 2. Basalrefers to basal medium as described in Example 2.

FIG. 18 is a graph showing that Bv8, similar to EG-VEGF, increases whiteblood cell count in vivo. Cell counts were measured at 3 days (lightgray), 6 days (dark gray), or 12 days (open bar) after in vivointroduction of Bv8-expressing adenoviral vectors.

FIGS. 19A-D are graphs showing the results of real time quantitative PCRexpression analysis of Bv8/EG-VEGF receptor-1 and Bv8/EG-VEGF receptor-2in human and mouse derived B lymphocytes, CD4+ T lymphocytes, CD8+ Tlymphocytes, and Natural Killer cells. FIGS. 19A and B show the relativetranscript level of Bv8/EG-VEGF receptor-1 in human (FIG. 19A) and mouse(FIG. 19B) derived B lymphocytes, CD4+ T lymphocytes, CD8+ Tlymphocytes, and Natural Killer cells. FIGS. 19C and D show the relativetranscript level of Bv8/EG-VEGF receptor-2 in human (FIG. 19C) and mouse(FIG. 19C) derived B lymphocytes, CD4+ T lymphocytes, CD8+ Tlymphocytes, and Natural Killer cells.

FIG. 20 is a graph showing that Bv8 and EG-VEGF increases 3H-thymidineincorporation in mouse B lymphocytes ex vivo.

FIG. 21 is a graph showing that Bv8 and EG-VEGF increase 3H-thymidineincorporation in mouse CD4+ T lymphocytes ex vivo. The insert showsincorporation of 3H-thymidine in CD4+ T cells in the presence ofincreasing concentrations of EG-VEGF or Bv8.

FIGS. 22A-D are graphs showing that EG-VEGF induces cytokine productionin CD4+ T cells. EG-VEGF induced production of IL-2 and IFN-γ in CD4+ Tcells.

FIGS. 23A-E are graphs showing that Bv8 promotes hematopoietic recoveryin vivo after myelosuppression with 5-FU. Cells counts were measured 5days, 11 days, and 14 days after in vivo introduction of Bv8-expressingadenoviral vectors. Flt^(sel) refers to a VEGF mutant that selectivelybinds FLT1 receptor. KDR^(sel) refers to a VEGF mutant that selectivelybinds KDR receptor. Bv8 increased white blood cell count, granulocytecount, monocyte count, and platelet count after myelosuppression with5-FU.

FIG. 24 is a graph showing spleen-derived committed mononuclear cellcolony formation in vitro in the presence of various growth factorsfollowing myelosuppression with 5-FU. Spleen cells isolated from animalstreated with Bv8 contained a significantly higher number of myeloidprogenitor cells (CFU-GM) than the non-virus or LacZ treated controlmice.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

I. Definitions

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. See, e.g. Singleton et al.,Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley &Sons (New York, N.Y. 1994); Sambrook et al., Molecular Cloning, ALaboratory Manual, Cold Springs Harbor Press (Cold Springs Harbor, N.Y.1989). For purposes of the present invention, the following terms aredefined below.

The terms “Bv8” and “Bv8 polypeptide,” are used interchangeably herein,and refer to native sequence Bv8, Bv8 variants, and chimeric Bv8, eachof which is defined herein. Optionally, the Bv8 is not associated withnative glycosylation. “Native glycosylation” refers to the carbohydratemoieties that are covalently attached to Bv8 when it is produced inmammalian cells, particularly in the cells in which it is produced innature. Accordingly, human Bv8 produced in a non-human cell is anexample of Bv8 that may “not be associated with native glycosylation.”Bv8 may not be glycosylated at all, as in the case where it is producedin prokaryotes, e.g. E. coli.

Bv8 polynucleotide is RNA or DNA that encodes a Bv8 polypeptide, asdefined above, or which hybridizes to such DNA or RNA and remains stablybound to it under stringent hybridization conditions and is greater thanabout 10 nucleotides in length. Stringent conditions are those which (1)employ low ionic strength and high temperature for washing, for example,0.15 M NaCl/0.015 M sodium citrate/0.1% NaDodSO₄ at 50° C., or (2) useduring hybridization a denaturing agent such as formamide, for example,50% (vol/vol) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1%polyvinlypyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mMNaCl, 75 mM sodium citrate at 42° C.

A polynucleotide is operably linked when it is placed into a functionalrelationship with another nucleic acid sequence. Bv8 polynucleotide maybe operably linked with another nucleic acid sequence in a vector suchthat it may be expressed in a particular host organism. This may be doneby methods well known in the art. For example, DNA for a presequence ora secretory leader is operably linked to DNA for a polypeptide if it isexpressed as a preprotein that participates in the secretion of thepolypeptide; a promoter or enhancer is operably linked to a codingsequence if it affects the transcription of the sequence; or a ribosomebinding site is operably linked to a coding sequence if it is positionedso as to facilitate translation. Generally, “operably linked” means thatthe DNA sequences being linked are contiguous and, in the case of asecretory leader, contiguous and in reading phase. However, enhancers donot have to be contiguous. Linking is accomplished by ligation atconvenient restriction sites. If such sites do not exist, then syntheticoligonucleotide adapters or linkers are used in accord with conventionalpractice.

“Native sequence Bv8” comprises a polypeptide having the same amino acidsequence as Bv8 derived from nature, regardless of its mode ofpreparation. Thus, native sequence Bv8 can have the amino acid sequenceof naturally occurring human Bv8, murine Bv8, or Bv8 from any othermammalian species. For example a full-length native sequence human Bv8amino acid sequence is shown in FIG. 2 (SEQ ID NO: 2). A secondfull-length native sequence human Bv8 is shown in FIG. 4 (SEQ ID NO: 4).These two sequences are the result of the alternative splicing of anexon that encodes a canonical heparin binding domain. Thus the nativesequence human Bv8 whose amino acid sequence is shown in FIG. 2 (SEQ IDNO: 2) comprises a heparin binding domain, while the native sequence Bv8depicted in FIG. 4 (SEQ ID NO: 4) does not. A native sequence mouse Bv8amino acid sequence is shown in FIG. 6 (SEQ ID NO: 6). Human and murineBv8 sequences are also disclosed, for example, in Wechselberger et al.,1999, FEBS Lett., 462:177-181 and Li et al., 2001, Mol. Pharm.,59:692-698. Such native sequence Bv8 can be isolated from nature or canbe produced by recombinant and/or synthetic means. The term “nativesequence Bv8” specifically encompasses naturally occurring prepro, pro,and mature forms and truncated forms of Bv8, naturally occurring variantforms (e.g. alternatively spliced forms, such as that shown in FIG. 4(SEQ ID NO: 4)), and naturally occurring allelic variants. A preferrednative sequence Bv8 is a full-length native sequence human Bv8 as shownin FIG. 2 (SEQ ID NO: 2).

“Bv8 variants” are biologically active Bv8 polypeptides having an aminoacid sequence that differs from the sequence of a native sequence Bv8polypeptide, such as those shown in FIGS. 2, 4 and 6 (SEQ ID NOs:2, 4and 6) for human and murine Bv8, by virtue of an insertion, deletion,modification, and/or substitution of one or more amino acid residueswithin the native sequence. Bv8 variants generally have less than 100%sequence identity with a native sequence Bv8, such as the human Bv8 ofFIG. 2 (SEQ ID NO: 2). Ordinarily, however, a biologically active Bv8variant will have an amino acid sequence with at least about 70% aminoacid sequence identity with the amino acid sequence of a naturallyoccurring Bv8 such as the human Bv8 of FIG. 2 (SEQ ID NO: 2), preferablyat least about 75%, more preferably at least about 80%, even morepreferably at least about 85%, even more preferably at least about 90%,with increasing preference of at least about 95% to at least about 99%amino acid sequence identity, in 1% increments. The Bv8 variants includepeptide fragments of at least 5 amino acids that retain a biologicalactivity of the corresponding native sequence Bv8 polypeptide. Bv8variants also include Bv8 polypeptides wherein one or more amino acidresidues are added at the N- or C-terminus of, or within, a native Bv8sequence. Bv8 variants also include Bv8 polypeptides where a number ofamino acid residues are deleted and optionally substituted by one ormore amino acid residues. Bv8 variants also may be covalently modified,for example by substitution with a moiety other than a naturallyoccurring amino acid or by modifying an amino acid residue to produce anon-naturally occurring amino acid. Bv8 variants may comprise a heparinbinding domain.

As used herein, the terms “EG-VEGF” and “EG-VEGF polypeptide,” which areused interchangeably, refer to native sequence EG-VEGF, EG-VEGFvariants, and chimeric EG-VEGF, each of which is defined herein.Optionally, the EG-VEGF is not associated with native glycosylation.“Native glycosylation” refers to the carbohydrate moieties that arecovalently attached to EG-VEGF when it is produced in mammalian cells,particularly in the cells in which it is produced in nature.Accordingly, human EG-VEGF produced in a non-human cell is an example ofEG-VEGF that may “not be associated with native glycosylation.”Sometimes the EG-VEGF may not be glycosylated at all, as in the casewhere it is produced in prokaryotes, e.g. E. coli.

EG-VEGF polynucleotide is RNA or DNA that encodes a EG-VEGF polypeptide,as defined above, or which hybridizes to such DNA or RNA and remainsstably bound to it under stringent hybridization conditions and isgreater than about 10 nucleotides in length. Stringent conditions arethose which (1) employ low ionic strength and high temperature forwashing, for example, 0.15 M NaCl/0.015 M sodium citrate/0.1% NaDodSO₄at 50° C., or (2) use during hybridization a denaturing agent such asformamide, for example, 50% (vol/vol) formamide with 0.1% bovine serumalbumin/0.1% Ficoll/0.1% polyvinlypyrrolidone/50 mM sodium phosphatebuffer at pH 6.5 with 750 mM NaCl, 75 mM sodium citrate at 42° C.

A polynucleotide is operably linked when it is placed into a functionalrelationship with another nucleic acid sequence. EG-VEGF polynucleotidemay be operably linked with another nucleic acid sequence in a vectorsuch that it may be expressed in a particular host organism. This may bedone by methods well known in the art. For example, DNA for apresequence or a secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous and, in thecase of a secretory leader, contiguous and in reading phase. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,then synthetic oligonucleotide adapters or linkers are used in accordwith conventional practice.

“Native sequence EG-VEGF” comprises a polypeptide having the same aminoacid sequence as EG-VEGF derived from nature, regardless of its mode ofpreparation. Thus, native sequence EG-VEGF can have the amino acidsequence of naturally occurring human EG-VEGF, murine EG-VEGF, orEG-VEGF from any other mammalian species. In an embodiment, afull-length native sequence human EG-VEGF comprises an amino acidsequence of SEQ ID NO:8. In an embodiment, a native sequence mouseEG-VEGF amino comprises an acid sequence of SEQ ID NO:10. Human andmurine EG-VEGF sequences are also disclosed, for example, in LeCouter etal., 2001, Nature, 412:877-884.

Such native sequence EG-VEGF can be isolated from nature or can beproduced by recombinant and/or synthetic means. The term “nativesequence EG-VEGF” specifically encompasses naturally occurring prepro,pro, and mature forms and truncated forms of EG-VEGF, naturallyoccurring variant forms (e.g. alternatively spliced forms), andnaturally occurring allelic variants. A preferred native sequenceEG-VEGF is a full-length native sequence human EG-VEGF comprising anamino acid sequence of SEQ ID NO:8.

“EG-VEGF variants” are biologically active EG-VEGF polypeptides havingan amino acid sequence that differs from the sequence of a nativesequence EG-VEGF polypeptide, such as for human and murine EG-VEGF (SEQID NOs:8 and 10), by virtue of an insertion, deletion, modification,and/or substitution of one or more amino acid residues within the nativesequence. EG-VEGF variants generally have less than 100% sequenceidentity with a native sequence EG-VEGF. Ordinarily, however, abiologically active EG-VEGF variant will have an amino acid sequencewith at least about 70% amino acid sequence identity with the amino acidsequence of a naturally occurring EG-VEGF, preferably at least about75%, more preferably at least about 80%, even more preferably at leastabout 85%, even more preferably at least about 90%, with increasingpreference of at least about 95% to at least about 99% amino acidsequence identity, in 1% increments. The EG-VEGF variants includepeptide fragments of at least 5 amino acids that retain a biologicalactivity of the corresponding native sequence EG-VEGF polypeptide.EG-VEGF variants also include EG-VEGF polypeptides wherein one or moreamino acid residues are added at the N- or C-terminus of, or within, anative EG-VEGF sequence. EG-VEGF variants also include EG-VEGFpolypeptides where a number of amino acid residues are deleted andoptionally substituted by one or more amino acid residues. EG-VEGFvariants also may be covalently modified, for example by substitutionwith a moiety other than a naturally occurring amino acid or bymodifying an amino acid residue to produce a non-naturally occurringamino acid.

“Percent amino acid sequence identity” with respect to the Bv8 orEG-VEGF sequence is defined herein as the percentage of amino acidresidues in the candidate sequence that are identical with the residuesin the Bv8 or EG-VEGF sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. None of N-terminal, C-terminal, or internalextensions, deletions, or insertions into the candidate Bv8 or EG-VEGFsequence shall be construed as affecting sequence identity or homology.Methods and computer programs for the alignment are well known in theart. One such computer program is “ALIGN-2,” authored by Genentech,Inc., which has been filed with user documentation in the United StatesCopyright Office, Washington, D.C. 20559, where it is registered underU.S. Copyright Registration No. TXU510087.

A “chimeric EG-VEGF” molecule is a polypeptide comprising full-lengthEG-VEGF or one or more domains thereof fused or bonded to heterologouspolypeptide. The chimeric EG-VEGF molecule will generally share at leastone biological property in common with naturally occurring EG-VEGF. Anexample of a chimeric EG-VEGF molecule is one that is epitope tagged forpurification purposes. Another chimeric EG-VEGF molecule is a EG-VEGFimmunoadhesin.

The term “epitope-tagged” when used herein refers to a chimericpolypeptide comprising Bv8 or EG-VEGF fused to a “tag polypeptide”. Thetag polypeptide has enough residues to provide an epitope against whichan antibody can be made, yet is short enough such that it does notinterfere with biological activity of the Bv8. The tag polypeptidepreferably is fairly unique so that the antibody against it does notsubstantially cross-react with other epitopes. Suitable tag polypeptidesgenerally have at least six amino acid residues and usually betweenabout 8-50 amino acid residues (preferably between about 9-30 residues).Preferred are poly-histidine sequences, which bind nickel, allowingisolation of the tagged protein by Ni-NTA chromatography as described(See, e.g., Lindsay et al., 1996, Neuron, 17:571-574).

“Isolated ” means Bv8 or EG-VEGF that has been purified from a Bv8 orEG-VEGF source, or has been prepared by recombinant or synthetic methodsand purified. Purified Bv8 or EG-VEGF is substantially free of otherpolypeptides or peptides. “Substantially free” here means less thanabout 5%, preferably less than about 2%, more preferably less than about1%, even more preferably less than about 0.5%, most preferably less thanabout 0.1% contamination with other source proteins.

“Essentially pure” protein means a composition comprising at least about90% by weight of the protein, based on total weight of the composition,preferably at least about 95% by weight, more preferably at least about90% by weight, even more preferably at least about 95% by weight.“Essentially homogeneous” protein means a composition comprising atleast about 99% by weight of protein, based on total weight of thecomposition.

“Agonists” are molecules or compounds that have one or more of thebiological properties of native sequence Bv8 or EG-VEGF. These mayinclude, but are not limited to, small organic molecules, peptides, andagonist anti-Bv8 or anti-EG-VEGF antibodies.

The term “antagonist” is used in the broadest sense, and includes anymolecule that partially or fully blocks, inhibits, or neutralizes abiological activity of a native Bv8 or EG-VEGF polypeptide. Suitableantagonist molecules specifically include antagonist antibodies orantibody fragments, fragments or amino acid sequence variants of nativeBv8 or EG-VEGF polypeptides, soluble Bv8 or EG-VEGF receptors orfragments thereof, peptides, small organic molecules, etc. Methods foridentifying agonists or antagonists of a Bv8 and/or EG-VEGF polypeptidemay comprise contacting a Bv8 or EG-VEGF polypeptide with a candidateagonist or antagonist molecule and measuring a detectable change in oneor more biological activities normally associated with the Bv8 orEG-VEGF polypeptide.

“Active” or “activity” for the purposes herein refers to form(s) of Bv8or EG-VEGF which retain a biological and/or an immunological activity ofnative or naturally-occurring Bv8 or EG-VEGF, wherein “biological”activity refers to a biological function (either inhibitory orstimulatory) caused by a native or naturally-occurring Bv8 or EG-VEGFother than the ability to induce the production of an antibody againstan antigenic epitope possessed by a native or naturally-occurring Bv8 orEG-VEGF and an “immunological” activity refers to the ability to inducethe production of an antibody against an antigenic epitope possessed bya native or naturally-occurring Bv8 or EG-VEGF.

Thus, “biologically active” when used in conjunction with “Bv8”,“isolated Bv8”, an agonist of Bv8, “EG-VEGF”, “isolated EG-VEGF”, or anagonist of EG-VEGF means a Bv8 or EG-VEGF polypeptide that exhibits orshares an effector function of native sequence Bv8 or EG-VEGFrespectively. A principal effector function of Bv8 of EG-VEGF is itsability to stimulate the proliferation of endothelial cells. Even morepreferably, the biological activity is the ability to regulatehematopoiesis.

“Biological property” when used in conjunction with “Bv8” or “isolatedBv8” or an “agonist” of Bv8, means having an effector or antigenicfunction or activity that is directly or indirectly caused or performedby native sequence Bv8 (whether in its native or denaturedconformation). Effector functions include enhancement of proliferationof endothelial cells, induction of angiogenesis and/or regulation ofhematopoiesis.

“Biological property” when used in conjunction with “EG-VEGF”, “isolatedEG-VEGF”, an “agonist” of EG-VEGF, means having an effector or antigenicfunction or activity that is directly or indirectly caused or performedby native sequence EG-VEGF respectively (whether in its native ordenatured conformation). Effector functions include enhancement ofproliferation of endothelial cells, induction of angiogenesis and/orregulation of hematopoiesis.

“Bv8 receptor” is a molecule to which Bv8 binds and mediates thebiological properties of Bv8. Bv8 receptors may also bind and mediatethe biological properties of EG-VEGF. Therefore, the term “Bv8 receptor”includes within its meaning Bv8/EG-VEGF receptor-1 and Bv8/EG-VEGFreceptor-2 (LeCouter et al., 2003, Proc. Natl. Acad. Sci. USA,100:2685-2690; Lin et al., 2002, J. Biol. Chem., 277:19276-19280; Masudaet al., 2002, Biochem. Biophys. Res. Commun., 293:396-402).

“EG-VEGF receptor” is a molecule to which EG-VEGF binds and mediates thebiological properties of EG-VEGF. EG-VEGF receptors may also bind andmediate the biological properties of Bv8. Therefore, the term “EG-VEGFreceptor” includes within its meaning Bv8/EG-VEGF receptor-1 andBv8/EG-VEGF receptor-2 (LeCouter et al., 2003 Proc. Natl. Acad. Sci.USA, 100:2685-2690; Lin et al., 2002, J. Biol. Chem., 277:19276-19280;Masuda et al., 2002, Biochem. Biophys. Res. Commun., 293:396-402).

The term “antibody” herein is used in the broadest sense andspecifically covers human, non-human (e.g. murine) and humanizedmonoclonal antibodies (including full length monoclonal antibodies),polyclonal antibodies, multispecific antibodies (e.g., bispecificantibodies), and antibody fragments so long as they exhibit the desiredbiological activity.

“Antibodies” (Abs) and “immunoglobulins” (Igs) are glycoproteins havingthe same structural characteristics. While antibodies exhibit bindingspecificity to a specific antigen, immunoglobulins include bothantibodies and other antibody-like molecules that lack antigenspecificity. Polypeptides of the latter kind are, for example, producedat low levels by the lymph system and at increased levels by myelomas.

“Native antibodies” and “native immunoglobulins” are usuallyheterotetrameric glycoproteins of about 150,000 daltons, composed of twoidentical light (L) chains and two identical heavy (H) chains. Eachlight chain is linked to a heavy chain by one covalent disulfide bond,while the number of disulfide linkages varies among the heavy chains ofdifferent immunoglobulin isotypes. Each heavy and light chain also hasregularly spaced intra-chain disulfide bridges. Each heavy chain has atone end a variable domain (V_(H)) followed by a number of constantdomains. Each light chain has a variable domain at one end (V_(L)) and aconstant domain at its other end; the constant domain of the light chainis aligned with the first constant domain of the heavy chain, and thelight-chain variable domain is aligned with the variable domain of theheavy chain. Particular amino acid residues are believed to form aninterface between the light- and heavy-chain variable domains.

The term “variable” refers to the fact that certain portions of thevariable domains differ extensively in sequence among antibodies and areused in the binding and specificity of each particular antibody for itsparticular antigen. However, the variability is not evenly distributedthroughout the variable domains of antibodies. It is concentrated inthree segments called hypervariable regions both in the light chain andthe heavy chain variable domains. The more highly conserved portions ofvariable domains are called the framework region (FR). The variabledomains of native heavy and light chains each comprise four FRs (FR1,FR2, FR3 and FR4, respectively), largely adopting a β-sheetconfiguration, connected by three hypervariable regions, which formloops connecting, and in some cases forming part of, the β-sheetstructure. The hypervariable regions in each chain are held together inclose proximity by the FRs and, with the hypervariable regions from theother chain, contribute to the formation of the antigen-binding site ofantibodies (see Kabat et al., Sequences of Proteins of ImmunologicalInterest, 5th Ed. Public Health Service, National Institutes of Health,Bethesda, Md. (1991), pages 647-669). The constant domains are notinvolved directly in binding an antibody to an antigen, but exhibitvarious effector functions, such as participation of the antibody inantibody-dependent cellular toxicity.

The term “hypervariable region” when used herein refers to the aminoacid residues of an antibody which are responsible for antigen binding.The hypervariable region comprises amino acid residues from a“complementarity determining region” or “CDR” (i.e. residues 24-34 (L1),50-56 (L2) and 89-97 (L3) in the light chain variable domain and 31-35(H1), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain;Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed.Public Health Service, National Institutes of Health, Bethesda, Md.(1991)) and/or those residues from a “hypervariable loop” (i.e. residues26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light chain variable domainand 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variabledomain; Chothia and Lesk, 1987, J. Mol. Biol. 196:901-917). “Framework”or “FR” residues are those variable domain residues other than thehypervariable region residues as herein defined.

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fc” fragment, whose name reflects its ability tocrystallize readily. Pepsin treatment yields an F(ab′)₂ fragment thathas two antigen-combining sites and is still capable of cross-linkingantigen.

“Fv” is the minimum antibody fragment that contains a completeantigen-recognition and -binding site. This region consists of a dimerof one heavy chain and one light chain variable domain in tight,non-covalent association. It is in this configuration that the threehypervariable regions of each variable domain interact to define anantigen-binding site on the surface of the V_(H)-V_(L) dimer.Collectively, the six hypervariable regions confer antigen-bindingspecificity to the antibody. However, even a single variable domain (orhalf of an Fv comprising only three hypervariable regions specific foran antigen) has the ability to recognize and bind antigen, although at alower affinity than the entire binding site.

The Fab fragment also contains the constant domain of the light chainand the first constant domain (CH1) of the heavy chain. Fab′ fragmentsdiffer from Fab fragments by the addition of a few residues at thecarboxyl terminus of the heavy chain CH1 domain including one or morecysteine(s) from the antibody hinge region. Fab′-SH is the designationherein for Fab′ in which the cysteine residue(s) of the constant domainsbear a free thiol group. F(ab′)₂ antibody fragments originally wereproduced as pairs of Fab′ fragments which have hinge cysteines betweenthem. Other chemical couplings of antibody fragments are also known.

The “light chains” of antibodies (immunoglobulins) from any vertebratespecies can be assigned to one of two clearly distinct types, calledkappa (κ) and lambda (λ), based on the amino acid sequences of theirconstant domains.

Depending on the amino acid sequence of the constant domain of theirheavy chains, immunoglobulins can be assigned to different classes.There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, andIgM, and several of these may be further divided into subclasses(isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. Theheavy-chain constant domains that correspond to the different classes ofimmunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunitstructures and three-dimensional configurations of different classes ofimmunoglobulins are well known.

“Antibody fragments” comprise a portion of a full-length antibody,generally the antigen binding or variable domain thereof. Examples ofantibody fragments include Fab, Fab′, F(ab′)₂, and Fv fragments;diabodies; linear antibodies; single-chain antibody molecules; andmulti-specific antibodies formed from antibody fragments.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast toconventional (polyclonal) antibody preparations that typically includedifferent antibodies directed against different determinants (epitopes),each monoclonal antibody is directed against a single determinant on theantigen. The modifier “monoclonal” indicates the character of theantibody as being obtained from a substantially homogeneous populationof antibodies, and is not to be construed as requiring production of theantibody by any particular method. For example, the monoclonalantibodies to be used in accordance with the present invention may bemade by the hybridoma method first described by Kohler et al., 1975,Nature 256:495, or may be made by recombinant DNA methods (see, e.g.,U.S. Pat. No. 4,816,567). The “monoclonal antibodies” may also beisolated from phage antibody libraries using the techniques described inClackson et al., 1991, Nature 352:624-628 and Marks et al., 1991, J.Mol. Biol. 222:581-597, for example.

The monoclonal antibodies herein specifically include “chimeric”antibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, so long asthey exhibit the desired biological activity (U.S. Pat. No. 4,816,567;and Morrison et al., 1984, Proc. Natl. Acad. Sci. USA, 81:6851-6855).

“Humanized” forms of non-human (e.g., murine) antibodies are chimericantibodies that contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which hypervariable regionresidues of the recipient are replaced by hypervariable region residuesfrom a non-human species (donor antibody) such as mouse, rat, rabbit ornon-human primate having the desired specificity, affinity, andcapacity. In some instances, framework region (FR) residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Furthermore, humanized antibodies may comprise residues that are notfound in the recipient antibody or in the donor antibody. Thesemodifications are made to further refine antibody performance. Ingeneral, the humanized antibody will comprise substantially all of atleast one, and typically two, variable domains, in which all orsubstantially all of the hypervariable regions correspond to those of anon-human immunoglobulin and all or substantially all of the FRs arethose of a human immunoglobulin sequence. The FRs may optionally bethose of a consensus or modified consensus sequence, as described, forexample, in Carter et. al, U.S. Pat. No. 6,054,297. The humanizedantibody optionally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see Jones et al., 1986, Nature,321:522-525; Reichmann et al., 1988, Nature, 332:323-329; and Presta,1992, Curr. Op. Struct. Biol., 2:593-596.

“Single-chain Fv” or “sFv” antibody fragments comprise the V_(H) andV_(L) domains of antibody, wherein these domains are present in a singlepolypeptide chain. Generally, the Fv polypeptide further comprises apolypeptide linker between the V_(H) and V_(L) domains which enables thesFv to form the desired structure for antigen binding. For a review ofsFv see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol.113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315(1994).

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy chain variabledomain (V_(H)) connected to a light chain variable domain (V_(L)) in thesame polypeptide chain (V_(H)-V_(L)). By using a linker that is tooshort to allow pairing between the two domains on the same chain, thedomains are forced to pair with the complementary domains of anotherchain and create two antigen-binding sites. Diabodies are described morefully in, for example, EP 404,097; WO 93/11161; and Hollinger et al.,1993, Proc. Natl. Acad. Sci. USA, 90:6444-6448.

The expression “linear antibodies” when used throughout this applicationrefers to the antibodies described in Zapata et al., 1995, Protein Eng.,8(10):1057-1062. Briefly, these antibodies comprise a pair of tandem Fdsegments (V_(H)-C_(H)1-V_(H)-C_(H)1) which form a pair of antigenbinding regions. Linear antibodies can be bispecific or monospecific.

The term “epitope” is used to refer to binding sites for (monoclonal orpolyclonal) antibodies on protein antigens.

By “agonist antibody” is meant an antibody that is a Bv8 or EG-VEGFagonist and thus possesses one or more of the biological properties ofnative sequence Bv8 or EG-VEGF.

The term “Bv8 immunoadhesin” and “EG-VEGF immunoadhesin” is usedinterchangeably with the term “Bv8 immunoglobulin chimera” and “EG-VEGFimmunoglobulin chimera”, respectively, and refers to a chimeric moleculethat combines at least a portion of a Bv8 or EG-VEGF molecule (native orvariant) with an immunoglobulin sequence. The immunoglobulin sequencepreferably, but not necessarily, is an immunoglobulin constant domain.Immunoadhesins can possess many of the valuable chemical and biologicalproperties of human antibodies. Since immunoadhesins can be constructedfrom a human protein sequence with a desired specificity linked to anappropriate human immunoglobulin hinge and constant domain (Fc)sequence, the binding specificity of interest can be achieved usingentirely human components. Such immunoadhesins are minimally immunogenicto the patient, and are safe for chronic or repeated use.

Examples of homomultimeric immunoadhesins that have been described fortherapeutic use include the CD4-IgG immunoadhesin for blocking thebinding of HIV to cell-surface CD4. Data obtained from Phase I clinicaltrials, in which CD4-IgG was administered to pregnant women just beforedelivery, suggests that this immunoadhesin may be useful in theprevention of maternal-fetal transfer of HIV (Ashkenazi et al., 1993,Intern. Rev. Immunol, 10:219-227). An immunoadhesin that binds tumornecrosis factor (TNF) has also been developed. TNF is a proinflammatorycytokine that has been shown to be a major mediator of septic shock.Based on a mouse model of septic shock, a TNF receptor immunoadhesin hasshown promise as a candidate for clinical use in treating septic shock(Ashkenazi et al., 1991, Proc. Natl. Acad. Sci. USA, 88:10535-10539).ENBREL® (etanercept), an immunoadhesin comprising a TNF receptorsequence fused to an IgG Fc region, was approved by the U.S. Food andDrug Administration (FDA), on Nov. 2, 1998, for the treatment ofrheumatoid arthritis. The new expanded use of ENBREL® in the treatmentof rheumatoid arthritis was approved by FDA on Jun. 6, 2000. For recentinformation on TNF blockers, including ENBREL®, see Lovell et al., 2000,N. Engl. J. Med. 342: 763-169, and accompanying editorial on p 810-811;and Weinblatt et al., 1999, N. Engl. J. Med. 340: 253-259; reviewed inMaini and Taylor, 2000, Annu. Rev. Med. 51: 207-229.

If the two arms of the immunoadhesin structure have differentspecificities, the immunoadhesin is called a “bispecific immunoadhesin”by analogy to bispecific antibodies. Dietsch et al., 1993, J. Immunol.Methods, 162:123 describe such a bispecific immunoadhesin combining theextracellular domains of the adhesion molecules, E-selectin andP-selectin, each of which selectin is expressed in a different cell typein nature. Binding studies indicated that the bispecific immunoglobulinfusion protein so formed had an enhanced ability to bind to a myeloidcell line compared to the monospecific immunoadhesins from which it wasderived.

The term “heteroadhesin” is used interchangeably with the expression“chimeric heteromultimer adhesin” and refers to a complex of chimericmolecules (amino acid sequences) in which each chimeric moleculecombines a biologically active portion, such as the extracellular domainof each of the heteromultimeric receptor monomers, with amultimerization domain. The “multimerization domain” promotes stableinteraction of the chimeric molecules within the heteromultimer complex.The multimerization domains may interact via an immunoglobulin sequence,leucine zipper, a hydrophobic region, a hydrophilic region, or a freethiol that forms an intermolecular disulfide bond between the chimericmolecules of the chimeric heteromultimer. The multimerization domain maycomprise an immunoglobulin constant region. In addition amultimerization region may be engineered such that steric interactionsnot only promote stable interaction, but further promote the formationof heterodimers over homodimers from a mixture of monomers.“Protuberances” are constructed by replacing small amino acid sidechains from the interface of the first polypeptide with larger sidechains (e.g. tyrosine or tryptophan). Compensatory “cavities” ofidentical or similar size to the protuberances are optionally created onthe interface of the second polypeptide by replacing large amino acidside chains with smaller ones (e.g. alanine or threonine). Theimmunoglobulin sequence preferably, but not necessarily, is animmunoglobulin constant domain. The immunoglobulin moiety in thechimeras of the present invention may be obtained from IgG₁, IgG₂, IgG₃or IgG₄ subtypes, IgA, IgE, IgD or IgM, but preferably IgG₁ or IgG₃.

As used herein, “treatment” is an approach for obtaining beneficial ordesired clinical results. For purposes of this invention, beneficial ordesired clinical results include, but are not limited to, alleviation ofsymptoms, diminishment of extent of disease, stabilized (i.e., notworsening) state of disease, delay or slowing of disease progression,amelioration or palliation of the disease state, and remission (whetherpartial or total), whether detectable or undetectable. “Treatment” canalso mean prolonging survival as compared to expected survival if notreceiving treatment. “Treatment” is an intervention performed with theintention of preventing the development or altering the pathology of adisorder. Accordingly, “treatment” refers to both therapeutic treatmentand prophylactic or preventative measures. Those in need of treatmentinclude those already with the disorder as well as those in which thedisorder is to be prevented. Specifically, the treatment may directlyprevent, slow down or otherwise decrease the pathology of cellulardegeneration or damage, such as the pathology of tumor cells in cancertreatment, or may render the cells more susceptible to treatment byother therapeutic agents.

“Chronic” administration refers to administration of the agent(s) in acontinuous mode as opposed to an acute mode, so as to maintain theinitial therapeutic effect (activity) for an extended period of time.“Intermittent” administration is treatment that is not consecutivelydone without interruption, but rather is cyclic in nature.

“Mammal” for purposes of treatment refers to any animal classified as amammal, including humans, other higher primates, domestic and farmanimals, and zoo, sports, or pet animals, such as dogs, cats, cattle,horses, sheep, pigs, goats, rabbits, and the like. Preferably, themammal is human.

“Tumor”, as used herein, refers to all neoplastic cell growth andproliferation, whether malignant or benign, and all pre-cancerous andcancerous cells and tissues.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. Examples of cancer include but are not limitedto, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. Moreparticular examples of such cancers include squamous cell cancer,small-cell lung cancer, non-small cell lung cancer, adenocarcinoma ofthe lung, squamous carcinoma of the lung, cancer of the peritoneum,hepatocellular cancer, gastrointestinal cancer, pancreatic cancer,glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladdercancer, hepatoma, breast cancer, colon cancer, colorectal cancer,endometrial or uterine carcinoma, salivary gland carcinoma, kidneycancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer,hepatic carcinoma, and various types of head and neck cancer.

“Hematological disorders” refers to disorders characterized by abnormalproliferation and/or differentiation of blood cells that can lead todysplastic changes in blood cells and hematologic malignancies. Manyhematological disorders can be classified as leukemias,myeloproliferative disorders (MPDs), myelodysplastic disorders,lymphoproliferative disorders, and lymphodysplastic disorders. Many ofthese disorders can occur in adults as well as children. Examples ofhematological disorders include, but are not limited to, acute myeloidleukemia (AML), chronic myelogenous leukemia (CML), acute lymphoblasticleukemia (ALL), multiple myeloma, T-cell lymphoma, lymphodysplasticleukemia, polycythaemia vera (PV), essential thrombocythaemia (ET), andmyeloid metaplasia (myelofibrosis).

The term “neutropenia” refers to disorders or conditions characterizedby an abnormally low or reduced number of circulating neutrophils.Neutropenia may be the result of disease, genetic disorders, drugs,toxins, radiation, and many therapeutic treatments, such as high dosechemotherapy and conventional oncology therapy. Examples of disorders orconditions associated with neutropenia include, but are not limited to,hematological disorders, infectious diseases including tuberculosis,typhoid, hepatitis, sepsis, acute bacterial disease, severemycobacterial or fungal disease, and mononucleosis, administration ofmyelotoxic and immunosuppressive agents including radiation,immunosuppressive drugs, and corticosteroids, cytotoxic chemotherapy orradiation therapy, infiltrative and hematological disorders includingleukemia, myeloma, Hodgkin's disease and lymphoma, agranulocytosis andaplastic anemia, histocytosis and sarcoidosis, surgery and traumaincluding burns, splenectomy, and anesthesia, alcoholic cirrhosis,aging, anticonvulsant drugs, uremia, diabetes, vitamin and mineraldeficiencies, and malnutrition. A patient suffering from neutropenia isat substantial risk for infection and disease, as the diminished numberof neutrophils circulating in the blood substantially impairs theability of the patient to fight any invading microorganisms.

The term “immunodeficiency disorders” refers to disorders or conditionscharacterized by a reduced or absent immune response. B cells, T cells,phagocytic cells, or complement may be deficient. The immunodeficiencydisorder may be primary or secondary. Leukopenias, includinglymphopenia, neutropenia, monocytopenia, and granulocytopenia, may beassociated with primary or secondary immunodeficiency disorders.Examples of primary immunodeficiency disorders include, but are notlimited to, B-cell deficiencies, including agammaglobulinemia andimmunoglobulin deficiency (Ig) with hyper-IgM, T cell deficienciesincluding DiGeorge anomaly, chronic mucocutaneous candidiasis, combinedimmunodeficiency with Igs, nucleoside phophorlyase, and idiopathic CD4lymphopenia, and combined T and B cell deficiencies including severecombined immunodeficiency, Wiskott-Aldrich syndrome, and X-linkedlymphoproliferative syndrome. Examples of secondary immunodeficiencydisorders include, but are not limited to, conditions associated withinfectious diseases including human acquired immunodeficiency virus(HIV), hepatitis, influenza, tuberculosis, typhoid, sepsis,cytomegalovirus, acute bacterial disease, severe mycobacterial or fungaldisease, congenital rubella, infectious mononucleosis, and viralexanthms, administration of myelotoxic and immunosuppressive agentsincluding radiation, immunosuppressive drugs, and corticosteroids,cytotoxic chemotherapy or radiation therapy, infiltrative andhematological disorders including leukemia, myeloma, Hodgkin's diseaseand lymphoma, agranulocytosis and aplastic anemia, histocytosis, andsarcoidosis, surgery and trauma including burns, splenectomy, andanesthesia, alcoholic cirrhosis, aging, anticonvulsant drugs, graft vs.host disease, uremia, diabetes, vitamin and mineral deficiencies, andmalnutrition.

The term “autoimmune disorder” refers to disorders mediated by sustainedadaptive immune responses to specific self antigens. Autoimmunedisorders may be categorized by the class of hypersensitivity responseassociated with the disorder. Types I-III are antibody-mediated. Type Iresponses are mediated by IgE, which induces mast cell activation. TypesII and III are mediated by IgG, which can activate eithercomplement-mediated or phagocytic effector mechanisms. Type II responsesare directed against cell-surface or matrix associated antigen leadingto tissue damage. Type III responses are directed against solubleantigens and the tissue damage is caused by downstream responsestriggered by immune complexes. Type IV responses are T lymphocytemediated and can be subdivided into two classes. In the first class,tissue damage is caused by inflammatory T cells (T_(H)1 cells) mediatedmainly by macrophages. In the second class, tissue damage is directlycaused by cytotoxic T cells. Examples of autoimmune disorders include,but are not limited to, graft versus host disease, inflammatory boweldiseases including Crohn's disease and colitis, Guillain-Barre'syndrome, lupus, multiple sclerosis, myasthenia gravis, optic neuritis,psoriasis, rheumatoid arthritis, Graves disease, autoimmune hepatitis,type I diabetes, aplastic anemia, interstitial cystitis, scleroderma,vulvodynia, neuromyotonia, and vitiligo.

The “pathology” of a disease includes all phenomena that compromise thewell-being of the patient. For cancer, this includes, withoutlimitation, abnormal or uncontrollable cell growth, metastasis,interference with the normal functioning of neighboring cells, releaseof cytokines or other secretory products at abnormal levels, suppressionor aggravation of inflammatory or immunological response, etc.

Administration “in combination with” one or more further therapeuticagents includes simultaneous (concurrent) and consecutive administrationin any order.

“Carriers” as used herein include pharmaceutically acceptable carriers,excipients, or stabilizers which are nontoxic to the cell or mammalbeing exposed thereto at the dosages and concentrations employed. Oftenthe physiologically acceptable carrier is an aqueous pH bufferedsolution. Examples of physiologically acceptable carriers includebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid; low molecular weight (less thanabout 10 residues) polypeptide; proteins, such as serum albumin,gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine, or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counterions such as sodium; and/or nonionic surfactantssuch as TWEEN™, polyethylene glycol (PEG), and PLURONICS™.

A “liposome” is a small vesicle composed of various types of lipids,phospholipids and/or surfactant which is useful for delivery of a drug(such as a Bv8 polypeptide or antibody thereto, an EG-VEGF polypeptideor antibody thereto, or combinations thereof) to a mammal. Thecomponents of the liposome are commonly arranged in a bilayer formation,similar to the lipid arrangement of biological membranes.

A “small molecule” is defined herein to have a molecular weight belowabout 500 Daltons.

The terms “vascular endothelial growth factor”, “VEGF”, “VEGFpolypeptide” and “VEGF protein” when used herein encompass nativesequence VEGF and VEGF variants (which are further defined herein). TheVEGF polypeptide may be isolated from a variety of sources, such as fromhuman tissue types or from another source, or prepared by recombinantand/or synthetic methods.

A “native sequence VEGF” comprises a polypeptide having the same aminoacid sequence as a VEGF derived from nature. Such native sequence VEGFcan be isolated from nature or can be produced by recombinant and/orsynthetic means. The term “native sequence VEGF” specificallyencompasses naturally-occurring truncated or secreted forms (e.g., anextracellular domain sequence), naturally-occurring variant forms (e.g.,alternatively spliced forms) and naturally-occurring allelic variants ofthe VEGF. In one embodiment of the invention, the native sequence VEGFis one of the five known isoforms, consisting of 121, 145, 165, 189, and206 amino acid residues, respectively, as described, for example in U.S.Pat. Nos. 5,332,671 and 5,240,848; in PCT Publication No. WO 98/10071;Leung et al., 1989, Science 246:1306-1309; and Keck et al., 1989,Science 246:1309-1312.

“VEGF variant polypeptide” means an active VEGF polypeptide as definedbelow having at least about 80%, preferably at least about 85%, morepreferably at least about 90%, event more preferably at least about 95%,most preferably at least about 98% amino acid sequence identity with theamino acid sequence of a native sequence VEGF. Such VEGF variantpolypeptides include, for instance, VEGF polypeptides wherein one ormore amino acid residues are added, or deleted, at the N- and/orC-terminus, as well as within one or more internal domains, of thenative sequence. In one embodiment of the invention, VEGF is a receptorspecific variant of native VEGF as described, for example, in PCTPublication Nos. WO 97/08313 and WO 00/63380 and U.S. Pat. No.6,020,473.

The sequence identity (either amino acid or nucleic acid) for VEGF isdetermined using the same approach specifically described with regard toBv8 or EG-VEGF. Similarly, the definitions provided for agonist andantagonists of Bv8 or EG-VEGF, including but not limited to antibodies,will apply to VEGF agonists and antagonists.

II. Methods for Carrying out the Invention

The present invention is based on the identification of novel expressionand activities of Bv8 and EG-VEGF in hematopoietic stem cells (HSCs),lineage-committed blood progenitor cells, and lymphocytes. Inparticular, as described in detail herein, Bv8, EG-VEGF, and theirreceptors are expressed in the HSCs in bone marrow, peripheral bloodleukocytes (PBLs) as well as many hematological malignant cell lines.Both in vitro and in vivo experiments showed that Bv8 and EG-VEGF arecapable of promoting colony formation of bone marrow mononuclear cellsand spleen-derived committed mononuclear progenitor cells, increasingpopulations of white blood cells, and promoting activation of Blymphocytes and T lymphocytes. Accordingly, Bv8 nucleic acids andpolypeptides, EG-VEGF nucleic acids and polypeptides, or combinationsthereof can be used in a number of assays and in diagnosis and treatmentof conditions associated with hematopoiesis, neutropenias,immunodeficiency disorders, and autoimmune disorders.

A. Hematopoiesis

Hematopoiesis refers to the proliferation and differentiation process,in which different types of blood cells develop from multipotent stemcells having the capacity to proliferate and differentiate. Most of theblood cells in the blood are short lived and thus need to be replacedconstantly throughout life. The levels of mature blood cells in thecirculation can change rapidly in response to different environmentalstress ranging from blood loss, infections, and the like. The major siteof hematopoiesis in humans, after about 20 weeks of fetal life, is thebone marrow (BM), a tissue consisting of a heterogeneous population ofcells including hematopoietic stem cells (HSCs), endothelial cells(ECs), and other stromal cells as well as cells involved in bonehomeostasis, including chondroclasts and osteoblasts. Gerber andFerrara, 2003, J. Mol. Med., 81:20-31.

Normal hematopoiesis is based on the dual functioning of multipotentstem cells. Extensive self-renewal maintains the population ofundifferentiated stem cells, whereas differentiation results in theformation of various types of mature blood cells that are grouped intoone of three major blood cell lineages: lymphoid, myeloid and erythroidcell lineages. The lymphoid lineage is comprised of B cells and T cells,which collectively function in antibody production and antigendetection, thereby functioning as a cellular and humoral immune system.The myeloid lineage is comprised of monocytes (macrophages),granulocytes (including neutrophils), and megakaryocytes, and monitorsthe bloodstream for antigens, scavenges antigens from the bloodstream,fights off infectious agents, and produces platelets that are involvedin blood clotting. The erythroid lineage is comprised of red blood cellsthat carry oxygen throughout the body.

The hematopoietic stem cells as well as committed progenitor cellsdestined to become neutrophils, erythrocytes, platelets, and the like,may be distinguished from most other cells by the presence of theparticular progenitor “marker” antigen that is present on the surface ofthese stem/progenitor cells. A group of antibodies capable ofrecognizing this particular marker antigen is referred to as “cluster ofdifferentiation 34” or “CD34”. The designation “CD34+” is used todescribe a cell as one that has the particular cell surface antigen thatis recognized by the CD34 group of antibodies. Stem cells, then, areCD34+. The majority of bone marrow cells that are CD34+, however, are Blymphocyte progenitor cells and myeloid progenitor cells.

1. Hematopoietic Factors

The development of early and differentiated hematopoietic cells isregulated by many hematopoietic growth factors, cytokines, andchemokines secreted by surrounding cells within the BM microenvironmentas well as various non-hematopoietic organs (e.g., liver, kidney) andnormal T lymphocytes. All these factors together modulate the biologicalfunctions and destinies of hematopoietic cells present in the BM.Janowska-Wieczorek et al., 2001, Stem Cells, 19:99-107.

At least four colony-stimulating factors (CSFs) are known to cooperatein the regulation of neutrophil production. These four factors, referredto as GM-CSF (granulocyte and macrophage), IL-3 (interleukin-3), G-CSF(granulocyte), and M-CSF (macrophage), that is also known as CSF-1, aresynthesized by macrophages, T cells, endothelial cells and other typesof cells. The potential of a progenitor cell to respond to a CSF isdetermined, in part, by the presence of receptors on the surface of thecell for that particular CSF and, in part, by the concentration of theparticular CSF. There also is some indication for indirect stimulation,whether via an accessory cell or by synergistic action with otherobligatory growth factors, such as c-kit ligand, IL-6 (interleukin-6),IL-11 (interleukin-11), IL-4 (interleukin-4), and IL-1 (interleukin-1).

2. Neutrophils

The main infection and disease-fighting cells of the human immune systemare the white blood cells (leukocytes), which are originated from themyeloid lineage and circulate through the blood system. Of the manytypes of leukocytes, the neutrophil, a subtype of granulocyte thatcontains an oddly shaped nucleus and a highly granulated cytoplasm, isthe most common cell type and accounts for about two thirds of theentire white blood cell population in humans. Neutrophils are mobile,responsive to chemotactic stimuli generated upon infection, and capableof moving into infected tissues to kill the invading microorganisms. Thekilling depends on the ability of the neutrophils to engulf themicroorganisms and to release oxygen radicals and microbicidal enzymes.Baggiolini, 1984, Experientia, 40:906-909.

Neutrophils differentiate from stem cells through a series ofintermediate precursor cells, which can be distinguished by theirmicroscopic morphological appearance, including such characteristics asthe size of their nuclei, the shape of their nuclei, cell size,nuclear/cytoplasmic ratio, presence/absence of granules, and stainingcharacteristics. Initially, the multipotent stem cell, which cannot bemeasured directly in vitro, gives rise to myeloid “progenitor cells”that generate precursors for all myeloid cell lines. The first myeloidprogenitor is designated CFU-GEMM for “colony forming unit—granulocyte,erythroid, macrophage and megakaryocyte”. The CFU-GEMM progenitor, inturn, will give rise to a CFU-GM progenitor cell, which is otherwiseknown as “colony forming unit—granulocyte and macrophage”. In all ofthese descriptive terms, “colony” refers to a cell that is capable ofgiving rise to more than 50 cells as measured in 14 day in vitro assaysfor clonal growth, under conditions as set forth in Example 5 of thepresent specification. These cells will divide at least six times.

The CFU-GM is a committed progenitor; it is committed to differentiatinginto granulocytes and macrophages only. It is not capable ofdifferentiating into other types of cells nor is it capable ofdedifferentiating into earlier stage progenitor cells. The CFU-GMprogenitor cell may then differentiate into a myeloblast. The timerequired for differentiation from a CFU-GEMM to a myeloblast is believedto be about 1-4 days. A myeloblast is the first of the series of cellsthat may be referred to as “precursors” to the neutrophils, as suchcells, once allowed to fully develop (differentiate), can only formneutrophils. Neutrophils are only capable of undergoing fewer than sixcell divisions and, therefore, do not form colonies in in vitro colonyassays as described previously.

Once differentiation has progressed to the myeloblast stage, themyeloblasts undergo terminal differentiation into promyelocytes, whichin turn differentiate into myelocytes over a course of about 4-6 days.Within another 5 days or so, myelocytes differentiate intometamyelocytes, which in turn differentiate into banded neutrophils.These banded neutrophils finally differentiate into mature, segmentedneutrophils, which have a half-life of about 0.3 to 2 days. The term“progenitor” will be used to refer to stem cells, and cells that canform colonies. “Precursor” will be used to refer to myeloblasts,promyelocytes and myelocytes and, in some instances, metamyelocytes andbanded neutrophils.

During this progressive, morphologic differentiation, changes in thesurface antigens of the precursor cells can be observed. For example,stem cells, CFU-GEMM and CFU-GM are CD34+. Hematopoietic cells thatdifferentiate beyond the CFU-GM stage are no longer CD34+. Similarprogressions of expression are observed for the cell-surface antigensCD33 and CD45RA. All neutrophil precursor cells subsequent to thepromyelocyte precursor cells may be characterized as CD34−, CD33+,CD38+, CD13+, CD45RA−, and CD15+. More mature cells also may becharacterized as CD11+ and CD16+ (Terstappen et. al., 1990, Leukemia,4:657). It should be appreciated, however, that such transitions incell-surface antigen expression are gradual, rather than abrupt, whereinsome cells of a particular precursor cell type may be positive and othercells of the same type may be negative for a particular cell-surfaceantigen. Furthermore, the determination that a particular cell type ispositive or negative for a particular cell-surface antigen will depend,in part, upon the particular method used to make that determination. Thecharacterization of cell differentiation by cell-surface antigenexpression may be confirmed by other means of characterizing celldifferentiation, such as cell morphology.

“Neutropenia” is a condition characterized by an abnormally low numberof circulating neutrophils. A patient suffering from neutropenia is atsubstantial risk for infection and disease, as the diminished number ofneutrophils circulating in the blood substantially impairs the abilityof the patient to fight any invading microorganisms. Neutropenia itselfmay be the result of disease, genetic disorders, drugs, toxins, andradiation as well as many therapeutic treatments, such as high dosechemotherapy (HDC) and conventional oncology therapy. For example,although many cancers have been found to be sensitive to extremely highdoses of radiation or anti-neoplastic (anti-cancer) drugs, suchintensive HDC is not widely used because it not only kills cancerouscells, but also frequently destroys the cells of the hematopoieticsystem that are responsible for generating the army of neutrophils thatare necessary to maintain a functioning immune system. Completedestruction of neutrophil progenitor and precursor cells eliminates thepatient's short-term capacity to generate mature neutrophils, therebyseverely compromising the patient's ability to combat infection. Thepatient then becomes “immunocompromised” and subject to opportunisticinfection. Such a condition may ultimately result in morbidity anddeath. Other situations also may be encountered where there has been asevere insult to the hematopoietic system, resulting in a substantialreduction in neutrophils and precursors thereto.

3. Hematological Disorders

Hematological disorders are characterized by abnormal proliferation anddifferentiation of blood cells, which can lead to dysplastic changes inblood cells and hematologic malignancies. Development of manyhematological disorders is a clonal process, in which one cell typepredominates. In some instances, other cell types also developabnormally. Furthermore, the abnormal cells of a particular disorderrepresent clonal derivatives of undifferentiated hematopoieticprogenitors, either multipotent stem cells or lineage-committedprecursor cells. Gerber and Ferrara, 2003, J. Mol. Med., 81:20-31;Raskind et al., 1998, Leukemia, 12:108-116. Many hematological disorderscan be classified as leukemias, myeloproliferative disorders (MPDs) andmyelodysplastic disorders. Many of these disorders can occur in adultsas well as children.

Acute myeloid leukemia (AML) is the most common type of acute leukemiathat occurs in adults. Several inherited genetic disorders andimmunodeficiency states are associated with an increased risk of AML.These include disorders with defects in DNA stability, leading to randomchromosomal breakage, such as Bloom's syndrome, Fanconi's anemia,Li-Fraumeni kindreds, ataxia-telangiectasia, and X-linkedagammaglobulinemia. Cytoarabine (Ara-C) has been used alone or incombination with anthracycline or daunorubicin to treat AML.

Acute lymphoblastic leukemia (ALL) is a heterogeneous disease withdistinct clinical features displayed by various subtypes. Reoccurringcytogenetic abnormalities have been demonstrated in ALL. The most commoncytogenetic abnormality involves translocation of chromosome 9 and 22.The resultant Philadelphia chromosome represents poor prognosis for thepatient. Vincristine, anthracyclines, and prednisone have been used totreat ALL.

The myeloproliferative disorders (MPD) are characterized by abnormalproliferation of hematopoietic cells. In each specific MPD, oneparticular cell type predominates, but there is evidence that some orall the other BM cell types are also proliferating abnormally to alesser extent. For example, chronic myelogenous leukemia (CML) is aclonal MPD of a pluripotent stem cell. CML is characterized by thepresence of large numbers of abnormal mature granulocytes, circulatingin the blood, due to a specific chromosomal abnormality involving thetranslocation of chromosomes 9 and 22, creating the Philadelphiachromosome. Ionizing radiation is associated with the development ofCML. Hydroxyurea, interferon (INF) and Ara-C have been used to treatpatients with CML. Other typical MPDs include, but not limited to,polycythemia vera (PV; over proliferation of red blood cells), essentialthrombocythemia (ET; over proliferation of platelets) and myeloidmetaplasia (also known as myelofibrosis).

The myelodysplastic syndromes (MDS) are heterogeneous clonalhematopoietic stem cell disorders grouped together because of thepresence of dysplastic changes in one or more of the hematopoieticlineages including dysplastic changes in the myeloid, erythroid, andmegakaryocytic series. These changes result in cytopenias in one or moreof the three lineages. Patients afflicted with MDS typically developcomplications related to anemia, neutropenia (infections), orthrombocytopenia (bleeding). Generally, from about 10% to about 70% ofpatients with MDS develop acute leukemia.

B. Bv8 and EG-VEGF

Bv8 is a small protein that was originally isolated from the skinsecretions of the frog Bombina variegata (Mollay et al., 1999, Eur. J.Pharmacol., 374:189-196). Bv8 belongs to a structurally related class ofpeptides including the digestive enzyme colipase, the Xenopushead-organizer, Dickkopf (Glinka et al., 1998, Nature, 391:357-362),venom protein A (VPRA) (Joubert and Strydom, 1980, Hopper-Seyler's Z.Physiol. Chem., 361:1787-1794) or MIT-1 (Schweitz et al. 1999, FEBSLett., 461:183-188), a nontoxic component of Dendroaspis polylepispolylepis venom, and the recently identified endocrine-gland-derivedvascular endothelial growth factor (EG-VEGF) (LeCouter et al., Nature,412:877-884 (2001)). A distinguishing structural motif is acolipase-fold, where 10 cysteine residues form five disulfide bridgeswithin a conserved span. EG-VEGF (80% identical to VPRA) and VPRA aremost closely related to the Bv8 peptide, with 83% and 79% identity,respectively. Mouse and human orthologues of Bv8, also known asprokineticin-2 (PK2) (Li et al., 2001, Mol. Pharm., 59:692-698), havebeen recently identified, and a variety of activities for these proteinshave been reported, including effects on neuronal survival,gastrointestinal smooth muscle contraction, and circadian locomotorrhythm. Li et al., 2001; Melchiorri et al., 2001, Eur. J. Neurosci.13:1694-1702; Cheng et al., 2002, Nature 417:405-410.

Both EG-VEGF and Bv8 have been identified as angiogenic factors withselective activities for endothelial cells of specific tissues. Thediverse structural and functional properties of endothelial cells, andevidence from a variety of in vivo and ex vivo systems, suggest theexistence of local, tissue-specific regulators of endothelial cellphenotype and growth. Expression of human (h)EG-VEGF mRNA was foundprincipally restricted to the steroidogenic glands: ovary, testis,adrenal, and placenta. EG-VEGF promoted proliferation, migration,survival, and fenestration in cultured adrenal capillary endothelialcells. It also induced extensive angiogenesis when delivered to theovary, but not other tissues. LeCouter et al., 2001, Nature,412:877-884.

Bv8 has been found expressed predominantly in the testis and is largelyrestricted to primary spermatocytes (LeCouter et al., 2003, PNAS5:2685-2690. Like EG-VEGF, Bv8 is able to induce proliferation,survival, and migration of adrenal cortical capillary endothelial cells.Bv8 gene expression is induced by hypoxic stress. Adenoviral delivery ofBv8 or EG-VEGF to mouse testis resulted in a potent angiogenic response.Furthermore, expression within the testis of two G protein-coupledreceptors for Bv8/EG-VEGF, Bv8/EG-VEGF receptor-1 and Bv8/EG-VEGFreceptor-2, was localized to vascular endothelial cells (LeCouter etal., 2003, Proc. Natl. Acad. Sci. USA, 100:2685-2690; Lin et al., 2002,J. Biol. Chem. 277:19276-19280; Masuda et al., 2002, Biochem. Biophys.Res. Commun. 293:396-402). The testis exhibits relatively high turnoverof endothelial cells. Thus, Bv8 and EG-VEGF, along with other factorssuch as VEGF, are considered to be important in maintaining theintegrity and regulating proliferation of the blood vessels in thetestis.

C. Identification of Bv8 and EG-VEGF Variants

In addition to the full-length native sequence Bv8 and EG-VEGFpolypeptides described herein, it is contemplated that Bv8 and EG-VEGFvariants can be identified, prepared and used in the present invention.Bv8 and EG-VEGF variants can be prepared by introducing appropriatenucleotide changes into the Bv8 or EG-VEGF DNA, and/or by synthesis ofthe desired Bv8 or EG-VEGF polypeptide. Those skilled in the art willappreciate that amino acid changes may alter post-translationalprocesses of the Bv8 or EG-VEGF, such as changing the number or positionof glycosylation sites. Methods for producing Bv8 and EG-VEGF variantsare preferably the same as for producing native sequence Bv8 and EG-VEGFas described in detail below, substituting the polynucleotide encodingthe variant for the polynucleotide encoding the native sequence.

Polynucleotide molecules that encode Bv8 or EG-VEGF are used in themethods of the present invention. cDNAs encoding two full-lengthvariants of human Bv8 are provided in FIGS. 1 and 2 (SEQ ID NOS: 1 and2), and the corresponding deduced amino acid sequences are provided inFIGS. 2 and 4 (SEQ ID NOS: 2 and 4). A cDNA encoding mouse Bv8 isprovided in FIG. 5 (SEQ ID NO: 5) and the corresponding deduced aminoacid sequence is provided in FIG. 6 (SEQ ID NO: 6). cDNAs encoding twofull length native EG-VEGFs (SEQ ID NOS:7 and 9) and the correspondingdeduced amino acid sequences (SEQ ID NOS:8 and 10) are useful in themethods of the present invention. The polynucleotides used in thepresent invention can be obtained using standard techniques well knownto those skilled in the art such as, for example, hybridizationscreening and PCR methodology.

Any nucleotide sequence that encodes the amino acid sequence of Bv8 orEG-VEGF can be used to generate recombinant molecules that direct theexpression of Bv8 or EG-VEGF, respectively. The methods of the presentinvention may also utilize a fusion polynucleotide between a Bv8 orEG-VEGF coding sequence and a second coding sequence for a heterologousprotein.

In order to clone full length homologous cDNA sequences from any speciesencoding the entire Bv8 or EG-VEGF cDNA, or to clone family members orvariant forms such as allelic variants, labeled DNA probes made fromfragments corresponding to any part of the cDNA sequences disclosedherein may be used to screen a cDNA library derived from a cell ortissue type believed to express Bv8 or EG-VEGF. More specifically,oligonucleotides corresponding to either the 5′ or 3′ terminus of thecoding sequence may be used to obtain longer nucleotide sequences.

It may be necessary to screen multiple cDNA libraries from differenttissues to obtain a full-length cDNA. In the event that it is difficultto identify cDNA clones encoding the complete 5′ terminal coding region,an often encountered situation in cDNA cloning, the RACE (RapidAmplification of cDNA Ends) technique may be used. RACE is a provenPCR-based strategy for amplifying the 5′ end of incomplete cDNAs.5′-RACE-Ready RNA synthesized from human placenta containing a uniqueanchor sequence is commercially available (Clontech). To obtain the 5′end of the cDNA, PCR is carried out on 5′-RACE-Ready cDNA using theprovided anchor primer and the 3′ primer. A secondary PCR is thencarried out using the anchored primer and a nested 3′ primer accordingto the manufacturer's instructions. Once obtained, the full length cDNAsequence may be translated into amino acid sequence and examined forcertain landmarks such as a continuous open reading frame flanked bytranslation initiation and termination sites, a potential signalsequence and finally overall structural identity to the Bv8 and/orEG-VEGF sequences disclosed herein.

Alternatively, a labeled probe may be used to screen a genomic libraryderived from any organism of interest using appropriate stringentconditions as described infra.

Isolation of a Bv8 or EG-VEGF coding sequence or a homologous sequencemay be carried out by the polymerase chain reactions (PCR) using twodegenerate oligonucleotide primer pools designed on the basis of the Bv8or EG-VEGF coding sequences disclosed herein. The template for thereaction may be cDNA obtained by reverse transcription (RT) of mRNAprepared from, for example, human or non-human cell lines or tissuesknown or suspected to express a Bv8 gene allele or an EG-VEGF geneallele.

The PCR product may be subcloned and sequenced to ensure that theamplified sequences represent the sequences of a Bv8 or EG-VEGF codingsequence. The PCR fragment may then be used to isolate a full-lengthcDNA clone by a variety of methods. For example, the amplified fragmentmay be labeled and used to screen a bacteriophage cDNA library.Alternatively, the labeled fragment may be used to isolate genomicclones via the screening of a genomic library.

PCR technology may also be utilized to isolate full-length cDNAsequences. For example, RNA may be isolated, following standardprocedures, from an appropriate cellular or tissue source. An RTreaction may be performed on the RNA using an oligonucleotide primerspecific for the most 5′ end of the amplified fragment for the primingof first strand synthesis. The resulting RNA/DNA hybrid may then be“tailed” with guanines using a standard terminal transferase reaction,the hybrid may be digested with RNAase H, and second strand synthesismay then be primed with a poly-C primer. Thus, cDNA sequences upstreamof the amplified fragment may easily be isolated.

A cDNA clone of a mutant or allelic variant of the Bv8 or EG-VEGF genemay be isolated, for example, by using PCR. In this case, the first cDNAstrand may be synthesized by hybridizing an oligo-dT oligonucleotide tomRNA isolated from tissue known or suspected to express Bv8, EG-VEGF, ora combination thereof in an individual putatively carrying the mutantBv8 allele, mutant EG-VEGF allele, or combinations thereof, and byextending the new strand with reverse transcriptase. The second strandof the cDNA is then synthesized using an oligonucleotide that hybridizesspecifically to the 5′ end of the normal gene. Using these two primers,the product is then amplified via PCR, cloned into a suitable vector,and subjected to DNA sequence analysis through methods well known tothose of skill in the art. By comparing the DNA sequence of the mutantBv8 or EG-VEGF allele to that of the normal Bv8 or EG-VEGF allele, themutation(s) responsible for the loss or alteration of function of themutant Bv8 or EG-VEGF gene product can be ascertained.

Alternatively, a genomic library can be constructed using DNA obtainedfrom an individual suspected of or known to carry a mutant Bv8 allele ormutant EG-VEGF allele, or a cDNA library can be constructed using RNAfrom a tissue known, or suspected, to express a mutant Bv8 allele ormutant EG-VEGF allele. An unimpaired Bv8 or EG-VEGF gene or any suitablefragment thereof may then be labeled and used as a probe to identify thecorresponding mutant Bv8 or EG-VEGF allele in such libraries. Clonescontaining the mutant Bv8 gene sequences or mutant EG-VEGF genesequences may then be purified and subjected to sequence analysisaccording to methods well known to those of skill in the art.

Additionally, an expression library can be constructed utilizing cDNAsynthesized from, for example, RNA isolated from a tissue known, orsuspected, to express a mutant Bv8 allele or mutant EG-VEGF allele in anindividual suspected of or known to carry such a mutant allele. In thismanner, gene products made by the putatively mutant tissue may beexpressed and screened using standard antibody screening techniques inconjunction with antibodies raised against the normal Bv8 or EG-VEGFgene product, as described, below.

As used herein, the terms nucleic acid, polynucleotides and nucleotideare interchangeable, and refer to any nucleic acid, whether composed ofdeoxyribonucleosides or ribonucleosides, and whether composed ofphosphodiester linkages or modified linkages such as phosphotriester,phosphoramidate, siloxane, carbonate, carboxymethylester, acetamidate,carbamate, thioether, bridged phosphoramidate, bridged methylenephosphonate, bridged phosphoramidate, bridged phosphoramidate, bridgedmethylene phosphonate, phosphorothioate, methylphosphonate,phosphorodithioate, bridged phosphorothioate or sultone linkages, andcombinations of such linkages.

The terms nucleic acid, polynucleotide, and nucleotide also specificallyinclude nucleic acids composed of bases other than the five biologicallyoccurring bases (adenine, guanine, thymine, cytosine, and uracil). Forexample, a polynucleotide of the invention might contain at least onemodified base moiety which is selected from the group including, but notlimited to 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl -2-thiouridine,5-carboxymethylaminomethyl-uracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine,1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine,3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine,5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil,beta-D-mannosylqueosine, 5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine.

Furthermore, a polynucleotide used in the invention may comprise atleast one modified sugar moiety selected from the group including butnot limited to arabinose, 2-fluoroarabinose, xylulose, and hexose.

It is not intended that the methods of the present invention be limitedby the source of the polynucleotide. The polynucleotide can be from ahuman or non-human mammal, derived from any recombinant source,synthesized in vitro or by chemical synthesis. The nucleotide may be DNAor RNA and may exist in a double-stranded, single-stranded or partiallydouble-stranded form.

Nucleic acids useful in the present invention include, by way of exampleand not limitation, oligonucleotides such as antisense DNAs and/or RNAs;ribozymes; DNA for gene therapy; DNA and/or RNA chimeras; variousstructural forms of DNA including single-stranded DNA, double-strandedDNA, supercoiled DNA and/or triple-helix DNA; Z-DNA; and the like. Thenucleic acids may be prepared by any conventional means typically usedto prepare nucleic acids in large quantity. For example, DNAs and RNAsmay be chemically synthesized using commercially available reagents andsynthesizers by methods that are well-known in the art (see, e.g., Gait,1985, Oligonucleotide Synthesis: A Practical Approach, IRL Press,Oxford, England). RNAs may be produce in high yield via in vitrotranscription using plasmids such as SP65 (Promega Corporation, Madison,Wis.).

Any mRNA transcript encoded by Bv8 or EG-VEGF nucleic acid sequences maybe used in the methods of the present invention, including, inparticular, mRNA transcripts resulting from alternative splicing orprocessing of mRNA precursors.

In some circumstances, such as where increased nuclease stability isdesired, nucleic acids having modified internucleoside linkages may bepreferred. Nucleic acids containing modified internucleoside linkagesmay also be synthesized using reagents and methods that are well knownin the art. For example, methods for synthesizing nucleic acidscontaining phosphonate phosphorothioate, phosphorodithioate,phosphoramidate methoxyethyl phosphoramidate, formacetal,thioformacetal, diisopropylsilyl, acetamidate, carbamate,dimethylene-sulfide (—CH₂—S—CH₂), dimethylene-sulfoxide (—CH₂—SO—CH₂),dimethylene-sulfone (—CH₂—SO₂—CH₂), 2′-O-alkyl, and 2′-deoxy-2′-fluorophosphorothioate internucleoside linkages are well known in the art (seeUhlmann et al., 1990, Chem. Rev., 90:543-584; Schneider et al., 1990,Tetrahedron Lett., 31:335 and references cited therein).

In some embodiments of the present invention, the nucleotide used is anα-anomeric nucleotide. An α-anomeric nucleotide forms specificdouble-stranded hybrids with complementary RNA in which, contrary to theusual β-units, the strands run parallel to each other (Gautier et al.,1987, Nucl. Acids Res. 15:6625-6641). The nucleotide is a2′-0-methylribonucleotide (Inoue et al., 1987, Nucl. Acids Res.15:6131-6148), or a chimeric RNA-DNA analogue (Inoue et al., 1987, FEBSLett. 215:327-330).

The nucleic acids may be purified by any suitable means, as are wellknown in the art. For example, the nucleic acids can be purified byreverse phase or ion exchange HPLC, size exclusion chromatography, orgel electrophoresis. Of course, the skilled artisan will recognize thatthe method of purification will depend in part on the size of the DNA tobe purified.

Isolated or purified polynucleotides having at least 10 nucleotides(i.e., a hybridizable portion) of a Bv8 coding sequence, EG-VEGF codingsequence, a combination thereof, or the complement thereof, may also beused in the methods of the present invention. In other embodiments, thepolynucleotides contain at least 25 (continuous) nucleotides, 50nucleotides, 100 nucleotides, 150 nucleotides, or 200 nucleotides of aBv8 coding sequence, EG-VEGF coding sequence, or a combination thereof,or a full-length Bv8 coding sequence, EG-VEGF coding sequence, or acombination thereof. Nucleic acids can be single or double stranded.Additionally, the invention provides to polynucleotides that selectivelyhybridize to a complement of the foregoing coding sequences. Inpreferred embodiments, the polynucleotides contain at least 10, 25, 50,100, 150 or 200 nucleotides, or the entire length of a Bv8 codingsequence, EG-VEGF coding sequence, or a combination thereof.

Nucleotide sequences that encode a mutant of Bv8 or EG-VEGF peptidefragments of Bv8 or EG-VEGF truncated forms of Bv8 or EG-VEGF, and Bv8fusion proteins or EG-VEGF fusion proteins may also be useful in themethods of the present invention. Nucleotides encoding fusion proteinsmay include, but are not limited to, full length Bv8 sequences orEG-VEGF sequences truncated forms of Bv8 or EG-VEGF or nucleotidesencoding peptide fragments of Bv8 or EG-VEGF fused to an unrelatedprotein or peptide, such as for example, a domain fused to an Ig Fcdomain that increases the stability and half life of the resultingfusion protein (for example, Bv8-Ig or EG-VEGF-Ig) in the bloodstream;or an enzyme such as a fluorescent protein or a luminescent protein thatcan be used as a marker.

Furthermore, Bv8 and EG-VEGF polynucleotide variants that have beengenerated, at least in part, by some form of directed evolution, forexample, gene shuffling and/or recursive sequence recombinationdescribed in U.S. Pat. Nos. 5,605,793 and 5,837,458, incorporated byreference herein in their entirety, may be used in the methods of thepresent invention. For example, using such techniques, a Bv8 encodingsequence and/or EG-VEGF encoding sequence, or a plurality of Bv8 and/orEG-VEGF encoding sequences can be used as the starting point for thegeneration of novel sequences encoding functionally and/or structurallysimilar proteins with altered functional and/or structuralcharacteristics.

Highly related gene homologs of the Bv8 and/or EG-VEGF encodingpolynucleotide sequences described above may also be useful in thepresent invention. Highly related gene homologs are polynucleotidesencoding proteins that have at least about 60% amino acid sequenceidentity with the amino acid sequence of a naturally occurring Bv8 orEG-VEGF such as the mature human Bv8 of FIG. 2 or FIG. 4 (SEQ ID NOs:2and 4), and mature human EG-VEGF (SEQ ID NO:8), preferably at leastabout 65%, 70%, 75%, or 80% amino acid sequence identity, withincreasing preference of at least about 85% to at least about 99% aminoacid sequence identity, in 1% increments. Highly related homologs canencode proteins sharing functional activities with Bv8 and/or EG-VEGF.

The methods of the present invention also benefit by the use of (a) DNAvectors that contain any of the foregoing Bv8 or EG-VEGF codingsequences and/or their complements (i.e., antisense); (b) DNA expressionvectors that contain any of the foregoing Bv8 or EG-VEGF codingsequences operatively associated with a regulatory element that directsthe expression of the coding sequences; (c) genetically engineered hostcells that contain any of the foregoing Bv8 and/or EG-VEGF codingsequences, or combinations thereof operatively associated with aregulatory element that directs the expression of the coding sequencesin the host cell; and (d) genetically engineered host cells that expressan endogenous Bv8 gene or EG-VEGF gene under the control of anexogenously introduced regulatory element (i.e., gene activation).

Variations in native sequence Bv8 or EG-VEGF or in various domains ofthe Bv8 or EG-VEGF described herein, can be made, for example, using anyof the techniques and guidelines for conservative and non-conservativemutations set forth, for instance, in U.S. Pat. No. 5,364,934.Variations may be a substitution, deletion or insertion of one or morecodons encoding Bv8 or EG-VEGF that results in a change in the aminoacid sequence of the Bv8 or EG-VEGF as compared with native sequence Bv8or EG-VEGF. Optionally the variation is by substitution of at least oneamino acid with any other amino acid in one or more of the domains ofthe Bv8 or EG-VEGF. Guidance in determining which amino acid residue maybe inserted, substituted or deleted without adversely affecting thedesired activity may be found by comparing the sequence of Bv8 orEG-VEGF with that of homologous known protein molecules and minimizingthe number of amino acid sequence changes made in regions of highhomology. Amino acid substitutions can be the result of replacing oneamino acid with another amino acid having similar structural and/orchemical properties, such as the replacement of a leucine with a serine,i.e., conservative amino acid replacements. Insertions or deletions mayoptionally be in the range of about 1 to 5 amino acids. The variationallowed may be determined by systematically making insertions, deletionsor substitutions of amino acids in the sequence and testing theresulting variants for activity exhibited by the full-length or maturenative sequence.

Bv8 polypeptide fragments and EG-VEGF polypeptide fragments are alsouseful in the methods of the present invention. Such fragments may betruncated at the N-terminus or C-terminus, or may lack internalresidues, for example, when compared with a full-length native protein.Certain fragments lack amino acid residues that are not essential for adesired biological activity of the Bv8 polypeptide or EG-VEGFpolypeptide.

Bv8 fragments and EG-VEGF fragments may be prepared by any of a numberof conventional techniques. Desired peptide fragments may be chemicallysynthesized. An alternative approach involves generating Bv8 or EG-VEGFfragments by enzymatic digestion, e.g., by treating the protein with anenzyme known to cleave proteins at sites defined by particular aminoacid residues, or by digesting the DNA with suitable restriction enzymesand isolating the desired fragment. Yet another suitable techniqueinvolves isolating and amplifying a DNA fragment encoding a desiredpolypeptide fragment by polymerase chain reaction (PCR).Oligonucleotides that define the desired termini of the DNA fragment areemployed at the 5′ and 3′ primers in the PCR. Preferably, Bv8 or EG-VEGFpolypeptide fragments share at least one biological and/or immunologicalactivity with a native Bv8 polypeptide and/or native EG-VEGFpolypeptide, respectively.

In particular embodiments, conservative substitutions of interest areshown in Table 1 under the heading of preferred substitutions. If suchsubstitutions result in a change in biological activity, then moresubstantial changes, denominated exemplary substitutions in Table 1, oras further described below in reference to amino acid classes, areintroduced and the products screened.

TABLE 1 Original Residue Exemplary Substitutions Preferred SubstitutionsAla (A) val; leu; ile val Arg (R) lys; gln; asn lys Asn (N) gln; his;lys; arg gln Asp (D) glu glu Cys (C) ser ser Gln (Q) asn asn Glu (E) aspasp Gly (G) pro; ala ala His (H) asn; gln; lys; arg arg Ile (I) leu;val; met; ala; phe; leu norleucine Leu (L) norleucine; ile; val ile met;ala; phe Lys (K) arg; gln; asn arg Met (M) leu; phe; ile leu Phe (F)leu; val; ile; ala; tyr leu Pro (P) ala ala Ser (S) thr thr Thr (T) serser Trp (W) tyr; phe tyr Tyr (Y) trp; phe; thr; ser phe Val (V) ile;leu; met; phe leu ala; norleucine

Substantial modifications in function or immunological identity of theBv8 polypeptide or EG-VEGF polypeptide are accomplished by selectingsubstitutions that differ significantly in their effect on maintaining(a) the structure of the polypeptide backbone in the area of thesubstitution, for example, as a sheet or helical conformation, (b) thecharge or hydrophobicity of the molecule at the target site, or (c) thebulk of the side chain. Naturally occurring residues are divided intogroups based on common side-chain properties:

-   (1) hydrophobic: norleucine, met, ala, val, leu, ile;-   (2) neutral hydrophilic: cys, ser, thr;-   (3) acidic: asp, glu;-   (4) basic: asn, gln, his, lys, arg;-   (5) residues that influence chain orientation: gly, pro; and-   (6) aromatic: trp, tyr, phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class. Such substituted residues also may beintroduced into the conservative substitution sites or, more preferably,into the remaining (non-conserved) sites.

The variations can be made using methods known in the art such asoligonucleotide-mediated (site-directed) mutagenesis, alanine scanning,and PCR mutagenesis. Site-directed mutagenesis (Carter et al., 1986,Nucl. Acids Res., 13:4331; Zoller et al., 1987, Nucl. Acids Res.,10:6487), cassette mutagenesis (Wells et al., 1985, Gene, 34:315),restriction selection mutagenesis (Wells et al., 1986, Philos. Trans. R.Soc. London SerA, 317:415) or other known techniques can be performed oncloned DNA to produce the Bv8 variant DNA and/or EG-VEGF variant DNA.

Scanning amino acid analysis can also be employed to identify one ormore amino acids along a contiguous sequence. Among the preferredscanning amino acids are relatively small, neutral amino acids. Suchamino acids include alanine, glycine, serine, and cysteine. Alanine istypically a preferred scanning amino acid among this group because iteliminates the side-chain beyond the beta-carbon and is less likely toalter the main-chain conformation of the variant (Cunningham and Wells,1989, Science, 244: 1081-1085). Alanine is also typically preferredbecause it is the most common amino acid. Further, it is frequentlyfound in both buried and exposed positions (Creighton, The Proteins,(W.H. Freeman & Co., N.Y.); Chothia, 1976, J. Mol. Biol., 150:1). Ifalanine substitution does not yield adequate amounts of variant, anisoteric amino acid can be used.

D. Production of Bv8, EG-VEGF, and Variants Thereof

Techniques suitable for the production of Bv8, EG-VEGF, and variantsthereof are well known in the art. Because the preferred techniques arethe same for native polypeptides and variants, the techniques describedbelow apply to Bv8 and EG-VEGF variants as well as to native sequenceBv8 and EG-VEGF, respectively.

The preferred methods of production include isolating Bv8 or EG-VEGFfrom an endogenous source of the polypeptide, peptide synthesis (using apeptide synthesizer), recombinant techniques, or any combination ofthese techniques.

Most of the discussion below pertains to recombinant production of Bv8or EG-VEGF by culturing cells transformed with a vector containing Bv8nucleic acid, EG-VEGF nucleic acid, or a combination thereof andrecovering the polypeptide from the cell culture. However, one of skillin the art will recognize that there are many ways of producing Bv8 andEG-VEGF.

Briefly, this method involves transforming primary human cellscontaining an Bv8-encoding gene or EG-VEGF encoding gene with aconstruct (i.e., vector) comprising an amplifiable gene (such asdihydrofolate reductase (DHFR) or others discussed below) and at leastone flanking region of a length of at least about 150 bp that ishomologous with a DNA sequence at the locus of the coding region of theBv8 gene or EG-VEGF gene to provide amplification of the Bv8 gene orEG-VEGF. The amplifiable gene must be at a site that does not interferewith expression of the Bv8 or EG-VEGF gene. The transformation isconducted such that the construct becomes homologously integrated intothe genome of the primary cells to define an amplifiable region.

Primary cells comprising the construct are then selected for by means ofthe amplifiable gene or other marker present in the construct. Thepresence of the marker gene establishes the presence and integration ofthe construct into the host genome. No further selection of the primarycells need be made, since selection will be made in the second host. Ifdesired, the occurrence of the homologous recombination event can bedetermined by employing PCR and either sequencing the resultingamplified DNA sequences or determining the appropriate length of the PCRfragment when DNA from correct homologous integrants is present andexpanding only those cells containing such fragments. Also if desired,the selected cells may be amplified at this point by stressing the cellswith the appropriate amplifying agent (such as methotrexate if theamplifiable gene is DHFR), so that multiple copies of the target geneare obtained. Preferably, however, the amplification step is notconducted until after the second transformation described below.

After the selection step, DNA portions of the genome, sufficiently largeto include the entire amplifiable region, are isolated from the selectedprimary cells. Secondary mammalian expression host cells are thentransformed with these genomic DNA portions and cloned, and clones areselected that contain the amplifiable region. The amplifiable region isthen amplified by means of an amplifying agent if not already amplifiedin the primary cells. Finally, the secondary expression host cells nowcomprising multiple copies of the amplifiable region containing Bv8 orEG-VEGF are grown so as to express the gene and produce the protein.

The DNA encoding Bv8 or EG-VEGF may be obtained from any cDNA libraryprepared from tissue believed to possess Bv8 mRNA or EG-VEGF mRNA,respectively, and to express it at a detectable level. Accordingly, Bv8or EG-VEGF DNA can be conveniently obtained from a cDNA libraryprepared, for example, from multiple human tissues. The Bv8-encodinggene or EG-VEGF-encoding gene may also be obtained from a genomiclibrary or by oligonucleotide synthesis.

Libraries are screened with probes (such as antibodies to Bv8 or EG-VEGFor oligonucleotides of about 20-80 bases) designed to identify the geneof interest or the protein encoded by it. Screening the cDNA or genomiclibrary with the selected probe may be conducted using standardprocedures as described in chapters 10-12 of Sambrook et al., MolecularCloning: A Laboratory Manual (New York: Cold Spring Harbor LaboratoryPress, 1989). An alternative means to isolate the gene encoding Bv8 orthe gene encoding EG-VEGF is to use PCR methodology as described insection 14 of Sambrook et al., supra.

A preferred method of isolating Bv8 and/or EG-VEGF cDNA is to usecarefully selected oligonucleotide sequences to screen cDNA librariesfrom various human tissues. The oligonucleotide sequences selected asprobes should be of sufficient length and sufficiently unambiguous thatfalse positives are minimized. Preferred sequences are obtained from thenaturally occurring Bv8 or EG-VEGF disclosed herein.

The oligonucleotide must be labeled such that it can be detected uponhybridization to DNA in the library being screened. The preferred methodof labeling is to use ³²P-labeled ATP with polynucleotide kinase, as iswell known in the art, to radiolabel the oligonucleotide. However, othermethods may be used to label the oligonucleotide, including, but notlimited to, biotinylation or enzyme labeling.

The nucleic acid (e.g., cDNA or genomic DNA) encoding Bv8 or EG-VEGF isinserted into a replicable vector for further cloning (amplification ofthe DNA) or for expression. Many vectors are available. The vectorcomponents generally include, but are not limited to, one or more of thefollowing: a signal sequence, an origin of replication, one or moremarker genes, an enhancer element, a promoter, and a transcriptiontermination sequence.

Bv8 or EG-VEGF useful in this invention may be produced recombinantlynot only directly, but also as a fusion polypeptide with a heterologouspolypeptide. The heterologous polypeptide is preferably a signalsequence or other polypeptide having a specific cleavage site at theN-terminus of the mature protein or polypeptide. In general, the signalsequence may be a component of the vector, or it may be a part of theBv8 DNA or EG-VEGF DNA that is inserted into the vector. Theheterologous signal sequence selected preferably is one that isrecognized and processed (i.e., cleaved by a signal peptidase) by thehost cell. For prokaryotic host cells that do not recognize and processthe native Bv8 signal sequence or EG-VEGF signal sequence, the signalsequence is substituted by a prokaryotic signal sequence selected, forexample, from the group consisting of the alkaline phosphatase,penicillinase, 1 pp, and heat-stable enterotoxin II leaders. For yeastsecretion the native signal sequence may be substituted by, e.g. theyeast invertase leader, α factor leader (including Saccharomyces andKluyveromyces α-factor leaders, the latter described in U.S. Pat. No.5,010,182 issued 23 Apr. 1991), or acid phosphatase leader, the C.albicans glucoamylase leader (EP 362,179 published 4 Apr. 1990), or thesignal described in WO 90/13646 published 15 Nov. 1990. For mammaliancell expression, the native signal sequence (e.g., the Bv8 or EG-VEGFpresequence that normally directs secretion of Bv8 or EG-VEGF from humancells in vivo) is satisfactory, although other mammalian signalsequences may be suitable, such as signal sequences from other animalBv8 or EG-VEGF polypeptides, and signal sequences from secretedpolypeptides of the same or related species, as well as viral secretoryleaders, for example, the herpes simplex gD signal.

The DNA for such precursor region is ligated in reading frame to DNAencoding the mature Bv8 or EG-VEGF, or a soluble variant thereof.

Both expression and cloning vectors contain a nucleic acid sequence thatenables the vector to replicate in one or more selected host cells.Generally, in cloning vectors this sequence is one that enables thevector to replicate independently of the host chromosomal DNA, andincludes origins of replication or autonomously replicating sequences.Such sequences are well known for a variety of bacteria, yeast, andviruses. The origin of replication from the plasmid pBR322 is suitablefor most Gram-negative bacteria, the 2μ plasmid origin is suitable foryeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV)are useful for cloning vectors in mammalian cells. Generally, the originof replication component is not needed for mammalian expression vectors(the SV40 origin may typically be used only because it contains theearly promoter).

Most expression vectors are “shuttle” vectors, that is, they are capableof replication in at least one class of organisms but can be transfectedinto another organism for expression. For example, a vector is cloned inE. coli and then the same vector is transfected into yeast or mammaliancells for expression even though it is not capable of replicatingindependently of the host cell chromosome.

DNA may also be amplified by insertion into the host genome. This isreadily accomplished using Bacillus species as hosts, for example, byincluding in the vector a DNA sequence that is complementary to asequence found in Bacillus genomic DNA. Transfection of Bacillus withthis vector results in homologous recombination with the genome andinsertion of Bv8 DNA and/or EG-VEGF DNA. However, the recovery ofgenomic DNA encoding Bv8 or EG-VEGF is more complex than that of anexogenously replicated vector because restriction enzyme digestion isrequired to excise the Bv8 and/or EG-VEGF DNA.

Expression and cloning vectors should contain a selection gene, alsotermed a selectable marker. This gene encodes a protein necessary forthe survival or growth of transformed host cells grown in a selectiveculture medium. Host cells not transformed with the vector containingthe selection gene will not survive in the culture medium. Typicalselection genes encode proteins that (a) confer resistance toantibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate,or tetracycline, (b) complement auxotrophic deficiencies, or (c) supplycritical nutrients not available from complex media, e.g., the geneencoding D-alanine racemase for Bacilli.

One example of a selection scheme utilizes a drug to arrest growth of ahost cell. Those cells that are successfully transformed with aheterologous gene produce a protein conferring drug resistance and thussurvive the selection regimen. Examples of such dominant selection usethe drugs neomycin, mycophenolic acid and hygromycin.

Another example of suitable selectable markers for mammalian cells arethose that enable the identification of cells competent to take up theBv8 or EG-VEGF nucleic acid, such as DHFR or thymidine kinase. Themammalian cell transformants are placed under selection pressure suchthat only the transformants are uniquely adapted to survive by virtue ofhaving taken up the marker. Selection pressure is imposed by culturingthe transformants under conditions in which the concentration ofselection agent in the medium is successively changed, thereby leadingto amplification of both the selection gene and the DNA that encodes Bv8and/or EG-VEGF. Amplification is the process by which genes in greaterdemand for the production of a protein critical for growth arereiterated in tandem within the chromosomes of successive generations ofrecombinant cells. Increased quantities of Bv8 and/or EG-VEGF aresynthesized from the amplified DNA. Other examples of amplifiable genesinclude metallothionein-I and -II, preferably primate metallothioneingenes, adenosine deaminase, ornithine decarboxylase, etc. A preferredvector system is provided in U.S. Pat. No. 5,561,053.

For example, cells transformed with the DHFR selection gene are firstidentified by culturing all of the transformants in a culture mediumthat contains methotrexate (Mtx), a competitive antagonist of DHFR. Anappropriate host cell when wild-type DHFR is employed is the Chinesehamster ovary (CHO) cell line deficient in DHFR activity, prepared andpropagated as described by Urlaub et al., Proc. Natl. Acad. Sci. USA,77:4216 (1980). The transformed cells are then exposed to increasedlevels of methotrexate. This leads to the synthesis of multiple copiesof the DHFR gene, and, concomitantly, multiple copies of other DNAcomprising the expression vectors, such as the DNA encoding Bv8 and/orEG-VEGF. This amplification technique can be used with any otherwisesuitable host, for example, ATCC No. CCL61 CHO-K1, notwithstanding thepresence of endogenous DHFR if, for example, a mutant DHFR gene that ishighly resistant to Mtx is employed (EP 117,060).

Alternatively, host cells (particularly wild-type hosts that containendogenous DHFR) transformed or co-transformed with DNA sequencesencoding Bv8 and/or EG-VEGF wild-type DHFR protein, and anotherselectable marker such as aminoglycoside 3′-phosphotransferase (APH) canbe selected by cell growth in medium containing a selection agent forthe selectable marker such as an aminoglycosidic antibiotic, e.g.,kanamycin, neomycin, or G418. See, for example, U.S. Pat. No. 4,965,199.

A suitable selection gene for use in yeast is the trp1 gene present inthe yeast plasmid YRp7 (Stinchcomb et al., 1979, Nature, 282:39). Thetrp1 gene provides a selection marker for a mutant strain of yeastlacking the ability to grow in tryptophan, for example, ATCC No. 44076or PEP4-1. Jones, 1977, Genetics, 85:12. The presence of the trp1 lesionin the yeast host cell genome then provides an effective environment fordetecting transformation by growth in the absence of tryptophan.Similarly, Leu2-deficient yeast strains (ATCC 20,622 or 38,626) arecomplemented by known plasmids bearing the Leu2 gene.

In addition, vectors derived from the 1.6 μm circular plasmid pKD1 canbe used for transformation of Kluyveromyces yeasts. Bianchi et al.,1987, Curr. Genet., 12:185. More recently, an expression system forlarge-scale production of recombinant calf chymosin was reported for K.lactis. Van den Berg, 1990, Bio/Technology, 8:135. Stable multi-copyexpression vectors for secretion of mature recombinant human serumalbumin by industrial strains of Kluyveromyces have also been disclosed.Fleer et al., 1991, Bio/Technology, 9:968-975.

Expression and cloning vectors usually contain a promoter that isrecognized by the host organism and is operably linked to the Bv8 and/orEG-VEGF nucleic acid. Promoters are untranslated sequences locatedupstream (5′) to the start codon of a structural gene (generally withinabout 100 to 1000 bp) that control the transcription and translation ofparticular nucleic acid sequence, such as the Bv8 and/or EG-VEGF nucleicacid sequence, to which they are operably linked. Such promoterstypically fall into two classes, inducible and constitutive. Induciblepromoters are promoters that initiate increased levels of transcriptionfrom DNA under their control in response to some change in cultureconditions, e.g. the presence or absence of a nutrient or a change intemperature. At this time a large number of promoters recognized by avariety of potential host cells are well known. These promoters areoperably linked to Bv8-encoding DNA and/or EG-VEGF encoding DNA byremoving the promoter from the source DNA by restriction enzymedigestion and inserting the isolated promoter sequence into the vector.The native Bv8 promoter sequence, native EG-VEGF promoter sequence, andmany heterologous promoters may be used to direct amplification and/orexpression of the Bv8 DNA or EG-VEGF DNA, respectively. However,heterologous promoters are preferred, as they generally permit greatertranscription and higher yields of Bv8 and/or EG-VEGF as compared to thenative promoter.

Promoters suitable for use with prokaryotic hosts include theβ-lactamase and lactose promoter systems (Chang et al., 1978, Nature,275:615; Goeddel et al., Nature, 281:544 (1979)), alkaline phosphatase,a tryptophan (trp) promoter system (Goeddel, Nucleic Acids Res., 8:4057(1980); EP 36,776), and hybrid promoters such as the tac promoter.deBoer et al., 1983, Proc. Natl. Acad. Sci. USA, 80:21-25. However,other known bacterial promoters are suitable. Their nucleotide sequenceshave been published, thereby enabling a skilled worker operably toligate them to DNA encoding Bv8 and/or EG-VEGF (Siebenlist et al., Cell,20:269 (1980)) using linkers or adaptors to supply any requiredrestriction sites. Promoters for use in bacterial systems also willcontain a Shine-Delgarno (S.D.) sequence operably linked to the DNAencoding Bv8 and/or EG-VEGF.

Promoter sequences are known for eukaryotes. Virtually all eukaryoticgenes have an AT-rich region located approximately 25 to 30 basesupstream from the site where transcription is initiated. Anothersequence found 70 to 80 bases upstream from the start of transcriptionof many genes is a CXCAAT region where X may be any nucleotide. At the3′ end of most eukaryotic genes is an AATAAA sequence that may be thesignal for addition of the poly-A tail to the 3′ end of the codingsequence. All of these sequences are suitably inserted into eukaryoticexpression vectors.

Examples of suitable promoting sequences for use with yeast hostsinclude the promoters for 3-phosphoglycerate kinase (Hitzeman et al.,1980, J. Biol. Chem., 255:2073) or other glycolytic enzymes (Hess etal., 1968, J. Adv. Enzyme Reg., 7:149; Holland, 1978, Biochemistry,17:4900), such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phospho-fructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase.

Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase,and enzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further described,for example, in EP 73,657. Yeast enhancers also are advantageously usedwith yeast promoters.

Bv8 transcription and/or EG-VEGF transcription from vectors in mammalianhost cells is controlled, for example, by promoters obtained from thegenomes of viruses such as polyoma virus, fowlpox virus (UK 2,211,504published 5 Jul. 1989), adenovirus (such as Adenovirus 5), bovinepapilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus,hepatitis-B virus, and most preferably Simian Virus 40 (SV40), fromheterologous mammalian promoters, e.g., the actin promoter or animmunoglobulin promoter, from heat-shock promoters, and from thepromoter normally associated with the Bv8 or EG-VEGF sequence, providedsuch promoters are compatible with the host cell systems.

The early and late promoters of the SV40 virus are conveniently obtainedas an SV40 restriction fragment that also contains the SV40 viral originof replication. Fiers et al., 1978, Nature, 273:113; Mulligan et al.,1980, Science, 209:1422-1427; Pavlakis et al., 1981, Proc. Natl. Acad.Sci. USA, 78:7398-7402. The immediate early promoter of the humancytomegalovirus is conveniently obtained as a HindIII E restrictionfragment. Greenaway et al., 1982, Gene, 18:355-360. A system forexpressing DNA in mammalian hosts using the bovine papilloma virus as avector is disclosed in U.S. Pat. No. 4,419,446. A modification of thissystem is described in U.S. Pat. No. 4,601,978. See also Gray et al.,1982, Nature, 295:503-508 on expressing cDNA encoding immune interferonin monkey cells; Reyes et al., 1982, Nature, 297:598-601 on expressionof human β-interferon cDNA in mouse cells under the control of athymidine kinase promoter from herpes simplex virus; Canaani et al.,Proc. 1982, Natl. Acad. Sci. USA, 79:5166-5170 on expression of thehuman interferon β1 gene in cultured mouse and rabbit cells; and Gormanet al., 1982, Proc. Natl. Acad. Sci. USA, 79:6777-6781 on expression ofbacterial CAT sequences in CV-1 monkey kidney cells, chicken embryofibroblasts, Chinese hamster ovary cells, HeLa cells, and mouse NIH-3T3cells using the Rous sarcoma virus long terminal repeat as a promoter.

Transcription of a DNA encoding Bv8 and/or EG-VEGF by higher eukaryotesis often increased by inserting an enhancer sequence into the vector.Enhancers are cis-acting elements of DNA, usually about from 10 to 300bp, that act on a promoter to increase its transcription. Enhancers arerelatively orientation and position independent, having been found 5′(Laimins et al., 1981, Proc. Natl. Acad. Sci. USA, 78:993) and 3′ (Luskyet al., 1983, Mol. Cell Bio., 3:1108) to the transcription unit, withinan intron (Banerji et al., 1983, Cell, 33:729), as well as within thecoding sequence itself. Osborne et al., 1984, Mol. Cell Bio., 4:1293.Many enhancer sequences are now known from mammalian genes (globin,elastase, albumin, α-fetoprotein, and insulin). Typically, however, onewill use an enhancer from a eukaryotic cell virus. Examples include theSV40 enhancer on the late side of the replication origin (bp 100-270),the cytomegalovirus early promoter enhancer, the polyoma enhancer on thelate side of the replication origin, and adenovirus enhancers. See alsoYaniv, 1982, Nature, 297:17-18 on enhancing elements for activation ofeukaryotic promoters. The enhancer may be spliced into the vector at aposition 5′ or 3′ to the Bv8-encoding sequence or EG-VEGF encodingsequence, but is preferably located at a site 5′ from the promoter.

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human, or nucleated cells from other multicellularorganisms) will also contain sequences necessary for the termination oftranscription and for stabilizing the mRNA. Such sequences are commonlyavailable from the 5′ and, occasionally 3′, untranslated regions ofeukaryotic or viral DNAs or cDNAs. These regions contain nucleotidesegments transcribed as polyadenylated fragments in the untranslatedportion of the mRNA encoding Bv8 and/or EG-VEGF.

Construction of suitable vectors containing one or more of theabove-listed components employs standard ligation techniques. Isolatedplasmids or DNA fragments are cleaved, tailored, and re-ligated in theform desired to generate the plasmids required.

For analysis to confirm correct sequences in plasmids constructed, theligation mixtures are used to transform E. coli K12 strain 294 (ATCC31,446) and successful transformants selected by ampicillin ortetracycline resistance where appropriate. Plasmids from thetransformants are prepared, analyzed by restriction endonucleasedigestion, and/or sequenced by the method of Messing et al., 1981,Nucleic Acids Res., 9:309 or by the method of Maxam et al., 1980,Methods in Enzymology, 65:499.

Particularly useful in the preparation of Bv8, EG-VEGF, and variantsthereof are expression vectors that provide for the transient expressionin mammalian cells of DNA encoding Bv8 and/or EG-VEGF. In general,transient expression involves the use of an expression vector that isable to replicate efficiently in a host cell, such that the host cellaccumulates many copies of the expression vector and, in turn,synthesizes high levels of a desired polypeptide encoded by theexpression vector. Sambrook et al., supra, pp. 16.17-16.22. Transientexpression systems, comprising a suitable expression vector and a hostcell, allow for the convenient positive identification of polypeptidesencoded by cloned DNAs, as well as for the rapid screening of suchpolypeptides for desired biological or physiological properties. Thus,transient expression systems are particularly useful in the inventionfor purposes of identifying analogs and variants of Bv8 and/or EG-VEGFthat are biologically active.

Other methods, vectors, and host cells suitable for adaptation to thesynthesis of Bv8, EG-VEGF, or a combination thereof in recombinantvertebrate cell culture are described in Gething et al., 1981, Nature,293:620-625; Mantei et al., 1979, Nature, 281:40-46; EP 117,060; and EP117,058. A particularly useful plasmid for mammalian cell cultureexpression of Bv8 and/or EG-VEGF is pRK5 (EP 307,247) or pSVI6B. WO91/08291 published 13 Jun. 1991.

Suitable host cells for cloning or expressing the DNA in the vectorsherein are prokaryote, yeast, or higher eukaryote cells. Suitableprokaryotes for this purpose include eubacteria, such as Gram-negativeor Gram-positive organisms, for example, Enterobacteriaceae such asEscherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus,Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratiamarcescans, and Shigella, as well as Bacilli such as B. subtilis and B.licheniformis (e.g., B. licheniformis 41P disclosed in DD 266,710published 12 Apr. 1989), Pseudomonas such as P. aeruginosa, andStreptomyces. One preferred E. coli cloning host is E. coli 294 (ATCC31,446), although other strains such as E. coli B, E. coli X1776 (ATCC31,537), and E. coli W3110 (ATCC 27,325) are suitable. These examplesare illustrative rather than limiting. Strain W3110 is a particularlypreferred host or parent host because it is a common host strain forrecombinant DNA product fermentations. Preferably, the host cell shouldsecrete minimal amounts of proteolytic enzymes. For example, strainW3110 may be modified to effect a genetic mutation in the genes encodingproteins, with examples of such hosts including E. coli W3110 strain27C7. The complete genotype of 27C7 is tonAΔ ptr3 phoAΔE15Δ(argF-lac)169 ompTΔ degP41 kan^(r). Strain 27C7 was deposited on 30Oct. 1991 in the American Type Culture Collection as ATCC No. 55,244.Alternatively, the strain of E. coli having mutant periplasmic proteasedisclosed in U.S. Pat. No. 4,946,783 issued 7 Aug. 1990 maybe employed.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts for Bv8-encodingvectors and/or EG-VEGF encoding vectors. Saccharomyces cerevisiae, orcommon baker's yeast, is the most commonly used among lower eukaryotichost microorganisms. However, a number of other genera, species, andstrains are commonly available and useful herein, such asSchizosaccharomyces pombe (Beach et al., 1981, Nature, 290:140; EP139,383 published 2 May 1985); Kluyveromyces hosts (U.S. Pat. No.4,943,529; Fleer et al., supra) such as, e.g., K. lactis (MW98-8C,CBS683, CBS4574; Louvencourt et al., 1983, J. Bacteriol., 737), K.fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906; Van denBerg et al., supra), K. thermotolerans, and K. marxianus; yarrowia (EP402,226); Pichia pastoris (EP 183,070; Sreekrishna et al., 1988, J.Basic Microbiol., 28:265-278); Candida; Trichoderma reesia (EP 244,234);Neurospora crassa (Case et al., 1979, Proc. Natl. Acad. Sci. USA,76:5259-5263); Schwanniomyces such as Schwanniomyces occidentalis (EP394,538 published 31 Oct. 1990); and filamentous fungi such as, e.g.,Neurospora, Penicillium, Tolypocladium (WO 91/00357 published 10 Jan.1991), and Aspergillus hosts such as A. nidulans (Ballance et al., 1983,Biochem. Biophys. Res. Commun., 112:284-289; Tilburn et al., 1983, Gene,26:205-221; Yelton et al., 1984, Proc. Natl. Acad. Sci. USA,81:1470-1474) and A. niger. Kelly et al, 1985, EMBO J., 4:475-479.

Suitable host cells for the expression of glycosylated Bv8 and/orglycosylated EG-VEGF are derived from multicellular organisms. Such hostcells are capable of complex processing and glycosylation activities. Inprinciple, any higher eukaryotic cell culture is workable, whether fromvertebrate or invertebrate culture. Examples of invertebrate cellsinclude plant and insect cells. Numerous baculoviral strains andvariants and corresponding permissive insect host cells from hosts suchas Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedesalbopictus (mosquito), Drosophila melanogaster (fruitfly), and Bombyxmori have been identified. See, e.g., Luckow et al., Bio/Technology,6:47-55 (1988); Miller et al., in Genetic Engineering, Setlow et al.,eds., Vol. 8 (Plenum Publishing, 1986), pp. 277-279; and Maeda et al.,1985, Nature, 315:592-594. A variety of viral strains for transfectionare publicly available, e.g., the L-1 variant of Autographa californicaNPV and the Bm-5 strain of Bombyx mori NPV, and such viruses may be usedas the virus herein according to the present invention, particularly fortransfection of Spodoptera frugiperda cells.

Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato,and tobacco can be utilized as hosts. Typically, plant cells aretransfected by incubation with certain strains of the bacteriumAgrobacterium tumefaciens that has been previously manipulated tocontain the Bv8-encoding DNA, EG-VEGF encoding DNA, or a combinationthereof. During incubation of the plant cell culture with A.tumefaciens, the DNA encoding the Bv8 and/or EG-VEGF is transferred tothe plant cell host such that it is transfected, and will, underappropriate conditions, express the Bv8-encoding DNA and/or EG-VEGFencoding DNA. In addition, regulatory and signal sequences compatiblewith plant cells are available, such as the nopaline synthase promoterand polyadenylation signal sequences. Depicker et al., 1982, J. Mol.Appl. Gen., 1:561. In addition, DNA segments isolated from the upstreamregion of the T-DNA 780 gene are capable of activating or increasingtranscription levels of plant-expressible genes in recombinantDNA-containing plant tissue. EP 321,196 published 21 Jun. 1989.

However, interest has been greatest in vertebrate cells, and propagationof vertebrate cells in culture (tissue culture) has become a routineprocedure. See, e.g., Tissue Culture, Academic Press, Kruse andPatterson, editors (1973). Examples of useful mammalian host cell linesare monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651);human embryonic kidney line (293 or 293 cells subcloned for growth insuspension culture, Graham et al., J. Gen Virol., 36:59 (1977)); babyhamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovarycells/-DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA, 77:4216(1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod., 23:243-251(1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkeykidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells(HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo ratliver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci.,383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line(Hep G2).

Host cells are transfected and preferably transformed with theabove-described expression or cloning vectors for Bv8 production and/orEG-VEGF production and cultured in conventional nutrient media modifiedas appropriate for inducing promoters, selecting transformants, oramplifying the genes encoding the desired sequences.

Transfection refers to the taking up of an expression vector by a hostcell whether or not any coding sequences are in fact expressed. Numerousmethods of transfection are known to the ordinarily skilled artisan, forexample, CaPO₄ and electroporation. Successful transfection is generallyrecognized when any indication of the operation of this vector occurswithin the host cell.

Transformation means introducing DNA into an organism so that the DNA isreplicable, either as an extrachromosomal element or by chromosomalintegrant. Depending on the host cell used, transformation is done usingstandard techniques appropriate to such cells. The calcium treatmentemploying calcium chloride, as described in section 1.82 of Sambrook etal., supra, or electroporation is generally used for prokaryotes orother cells that contain substantial cell-wall barriers. Infection withAgrobacterium tumefaciens is used for transformation of certain plantcells, as described by Shaw et al., 1983, Gene, 23:315 and WO 89/05859published 29 Jun. 1989. In addition, plants may be transfected usingultrasound treatment as described in WO 91/00358 published 10 Jan. 1991.

For mammalian cells without such cell walls, the calcium phosphateprecipitation method of Graham et al., 1978, Virology, 52:456-457 ispreferred. General aspects of mammalian cell host-system transformationshave been described in U.S. Pat. No. 4,399,216 issued 16 Aug. 1983.Transformations into yeast are typically carried out according to themethod of Van Solingen et al., 1977, J. Bact., 130:946 and Hsiao et al.,1979, Proc. Natl. Acad. Sci. USA, 76:3829. However, other methods forintroducing DNA into cells, such as by nuclear microinjection,electroporation, bacterial protoplast fusion with intact cells, orpolycations, e.g., polybrene, polyornithine, etc., may also be used. Forvarious techniques for transforming mammalian cells, see Keown et al.,1990, Methods in Enzymology, 185:527-537 and Mansour et al., 1988,Nature, 336:348-352.

Prokaryotic cells used to produce Bv8 polypeptide, EG-VEGF polypeptide,or a combination thereof, are cultured in suitable media as describedgenerally in Sambrook et al., supra.

The mammalian host cells used to produce the Bv8, EG-VEGF, or acombination thereof, of this invention may be cultured in a variety ofmedia. Commercially available media such as Ham's F10 (Sigma), MinimalEssential Medium ((MEM), Sigma), RPMI-1640 (Sigma), and Dulbecco'sModified Eagle's Medium ((DMEM), Sigma) are suitable for culturing thehost cells. In addition, any of the media described in Ham et al. 1979,Meth. Enz., 58:44, Barnes et al., 1980, Anal. Biochem., 102:255, U.S.Pat. Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO90/03430; WO 87/00195; or U.S. Pat. No. Re. 30,985 may be used asculture media for the host cells. Any of these media may be supplementedas necessary with hormones and/or other growth factors (such as insulin,transferrin, or epidermal growth factor), salts (such as sodiumchloride, calcium, magnesium, and phosphate), buffers (such as HEPES),nucleosides (such as adenosine and thymidine), antibiotics (such asGENTAMYCIN™ drug), trace elements (defined as inorganic compoundsusually present at final concentrations in the micromolar range), andglucose or an equivalent energy source. Any other necessary supplementsmay also be included at appropriate concentrations that would be knownto those skilled in the art. The culture conditions, such astemperature, pH, and the like, are those previously used with the hostcell selected for expression, and will be apparent to the ordinarilyskilled artisan.

In general, principles, protocols, and practical techniques formaximizing the productivity of mammalian cell cultures can be found inMammalian Cell Biotechnology: a Practical Approach, M. Butler, ed. (IRLPress, 1991).

The host cells referred to in this disclosure encompass cells in cultureas well as cells that are within a host animal.

Gene amplification and/or expression may be measured in a sampledirectly, for example, by conventional Southern blotting, Northernblotting to quantitate the transcription of mRNA (Thomas, 1980, Proc.Natl. Acad. Sci. USA, 77:5201-5205), dot blotting (DNA analysis), or insitu hybridization, using an appropriately labeled probe, based on thesequences provided herein. Various labels may be employed, most commonlyradioisotopes, particularly ³²P. However, other techniques may also beemployed, such as using biotin-modified nucleotides for introductioninto a polynucleotide. The biotin then serves as the site for binding toavidin or antibodies, which may be labeled with a wide variety oflabels, such as radionuclides, fluorescers, enzymes, or the like.Alternatively, antibodies may be employed that can recognize specificduplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybridduplexes or DNA-protein duplexes. The antibodies in turn may be labeledand the assay may be carried out where the duplex is bound to a surface,so that upon the formation of duplex on the surface, the presence ofantibody bound to the duplex can be detected.

Gene expression, alternatively, can be measured by immunologicalmethods, such as immunohistochemical staining of tissue sections andassay of cell culture or body fluids, to quantitate directly theexpression of gene product. With immunohistochemical stainingtechniques, a cell sample is prepared, typically by dehydration andfixation, followed by reaction with labeled antibodies specific for thegene product coupled, where the labels are usually visually detectable,such as enzymatic labels, fluorescent labels, luminescent labels, andthe like. A particularly sensitive staining technique suitable for usein the present invention is described by Hsu et al., 1980, Am. J. Clin.Path., 75:734-738.

Antibodies useful for immunohistochemical staining and/or assay ofsample fluids may be either monoclonal or polyclonal, and may beprepared as described herein.

Bv8 and/or EG-VEGF preferably is recovered from the culture medium as asecreted polypeptide, although it also may be recovered from host celllysates. If the Bv8 or EG-VEGF is membrane-bound, it can be releasedfrom the membrane using a suitable detergent solution (e.g. Triton-X100).

When Bv8, EG-VEGF, or a combination thereof is produced in a recombinantcell other than one of human origin, the Bv8 and/or EG-VEGF iscompletely free of proteins or polypeptides of human origin. However, itis necessary to purify Bv8 and/or EG-VEGF from recombinant cell proteinsor polypeptides to obtain preparations that are substantiallyhomogeneous as to Bv8 and/or EG-VEGF. As a first step, the culturemedium or lysate can be centrifuged to remove particulate cell debris.Bv8 and/or EG-VEGF can then be purified from contaminant solubleproteins and polypeptides with the following procedures, which areexemplary of suitable purification procedures: by fractionation on anion-exchange column; ethanol precipitation; reverse phase HPLC;chromatography on silica; chromatofocusing; immunoaffinity; epitope-tagbinding resin; SDS-PAGE; ammonium sulfate precipitation; gel filtrationusing, for example, Sephadex G-75; and protein A Sepharose columns toremove contaminants such as IgG.

E. Modifications of Bv8 and EG-VEGF

Covalent modifications of Bv8, EG-VEGF, and variants thereof areincluded within the scope of this invention. One type of covalentmodification includes reacting targeted amino acid residues of a Bv8polypeptide and/or EG-VEGF polypeptide with an organic derivatizingagent that is capable of reacting with selected side chains or the N- orC-terminal residues of the Bv8 or EG-VEGF. Derivatization withbifunctional agents is useful, for instance, for crosslinking Bv8 orEG-VEGF to a water-insoluble support matrix or surface for use in themethod for purifying anti-Bv8 antibodies or EG-VEGF antibodiesrespectively, and vice versa. Commonly used crosslinking agents include,e.g., 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylicacid, homobifunctional imidoesters, including disuccinimidyl esters suchas 3,3′-dithiobis(succinimidylpropionate), bifunctional maleimides suchas bis-N-maleimido-1,8-octane and agents such asmethyl-3-[(p-azidophenyl)dithio]propioimidate.

Other modifications include deamidation of glutaminyl and asparaginylresidues to the corresponding glutamyl and aspartyl residues,respectively, hydroxylation of proline and lysine, phosphorylation ofhydroxyl groups of seryl or threonyl residues, methylation of theα-amino groups of lysine, arginine, and histidine side chains (T. E.Creighton, 1983, Proteins: Structure and Molecular Properties, W.H.Freeman & Co., San Francisco, pp. 79-86), acetylation of the N-terminalamine, and amidation of any C-terminal carboxyl group.

Another type of covalent modification of the Bv8 polypeptide and/orEG-VEGF polypeptide included within the scope of this inventioncomprises altering the native glycosylation pattern of the polypeptide.“Altering the native glycosylation pattern” is intended for purposesherein to mean deleting one or more carbohydrate moieties found innative sequence Bv8 (either by removing the underlying glycosylationsite or by deleting the glycosylation by chemical and/or enzymaticmeans), and/or adding one or more glycosylation sites that are notpresent in the native sequence Bv8 or EG-VEGF native sequence. Inaddition, the phrase includes qualitative changes in the glycosylationof the native proteins, involving a change in the nature and proportionsof the various carbohydrate moieties present.

Addition of glycosylation sites to the Bv8 polypeptide or EG-VEGFpolypeptide may be accomplished by altering the amino acid sequence. Thealteration may be made, for example, by the addition of, or substitutionby, one or more serine or threonine residues to the native sequence Bv8or native sequence EG-VEGF (for O-linked glycosylation sites). The Bv8amino acid sequence or EG-VEGF amino acid sequence may optionally bealtered through changes at the DNA level, particularly by mutating theDNA encoding the Bv8 polypeptide or EG-VEGF polypeptide at pre-selectedbases such that codons are generated that will translate into thedesired amino acids.

Another means of increasing the number of carbohydrate moieties on theBv8 polypeptide or EG-VEGF polypeptide is by chemical or enzymaticcoupling of glycosides to the polypeptide. Such methods are described inthe art, e.g., in WO 87/05330 published 11 Sep. 1987, and in Aplin andWriston, 1981, CRC Crit. Rev. Biochem., pp. 259-306.

Removal of carbohydrate moieties present on the Bv8 polypeptide orEG-VEGF polypeptide may be accomplished chemically or enzymatically orby mutational substitution of codons encoding for amino acid residuesthat serve as targets for glycosylation. Chemical deglycosylationtechniques are known in the art and described, for instance, byHakimuddin, et al., 1987, Arch. Biochem. Biophys., 259:52 and by Edge etal., 1981, Anal. Biochem., 118:131. Enzymatic cleavage of carbohydratemoieties on polypeptides can be achieved by the use of a variety ofendo- and exo-glycosidases as described by Thotakura et al., 1987,Meth._Enzymol., 138:350.

Another type of covalent modification of Bv8 or EG-VEGF compriseslinking the Bv8 polypeptide or EG-VEGF polypeptide, respectively, to oneof a variety of nonproteinaceous polymers, e.g., polyethylene glycol(PEG), polypropylene glycol, or polyoxyalkylenes, in the manner setforth, for example, in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144;4,670,417; 4,791,192 or 4,179,337.

The Bv8 and EG-VEGF of the present invention may also be modified in away to form a chimeric molecule comprising Bv8 or EG-VEGF fused toanother, heterologous polypeptide or amino acid sequence.

In one embodiment, such a chimeric molecule comprises a fusion of theBv8 or EG-VEGF with a tag polypeptide that provides an epitope to whichan anti-tag antibody can selectively bind. The epitope tag is generallyplaced at the amino- or carboxyl-terminus of the Bv8 or EG-VEGF. Thepresence of such epitope-tagged forms of the Bv8 and/or EG-VEGF can bedetected using an antibody against the tag polypeptide. Also, provisionof the epitope tag enables the Bv8 and/or EG-VEGF to be readily purifiedby affinity purification using an anti-tag antibody or another type ofaffinity matrix that binds to the epitope tag. Various tag polypeptidesand their respective antibodies are well known in the art. Examplesinclude poly-histidine (poly-his) or poly-histidine-glycine(poly-his-gly) tags; the flue HA tag polypeptide and its antibody 12CA5(Field et al., 1988, Mol. Cell. Biol., 8:2159-2165); the c-myc tag andthe 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto (Evan et al.,1985, Molecular and Cellular Biology, 5:3610-3616); and the HerpesSimplex virus glycoprotein D (gD) tag and its antibody (Paborsky et al.,1990, Protein Engineering, 3(6):547-553). Other tag polypeptides includethe Flag-peptide (Hopp et al., 1988, BioTechnology, 6:1204-1210); theKT3 epitope peptide (Martin et al., 1992, Science, 255:192-194); anα-tubulin epitope peptide (Skinner et al., 1991, J. Biol. Chem.,266:15163-15166); and the T7 gene 10 protein peptide tag(Lutz-Freyermuth et al., 1990, Proc. Natl. Acad. Sci. USA,87:6393-6397).

In an alternative embodiment, the chimeric molecule may comprise afusion of Bv8 and/or EG-VEGF with an immunoglobulin or a particularregion of an immunoglobulin. For a bivalent form of the chimericmolecule (also referred to as an “immunoadhesin”), such a fusion couldbe to the Fc region of an IgG molecule.

The simplest and most straightforward immunoadhesin design combines thebinding region(s) of the “adhesin” protein with the hinge and Fc regionsof an immunoglobulin heavy chain. Ordinarily, when preparingBv8-immunoglobulin chimeras or EG-VEGF-immunoglobulin chimeras for usein the present invention, nucleic acid encoding Bv8 and/or EG-VEGF willbe fused C-terminally to nucleic acid encoding the N-terminus of animmunoglobulin constant domain sequence, however N-terminal fusions arealso possible.

Typically, in such fusions the encoded chimeric polypeptide will retainat least functionally active hinge and CH2 and CH3 domains of theconstant region of an immunoglobulin heavy chain. Fusions are also madeto the C-terminus of the Fc portion of a constant domain, or immediatelyN-terminal to the CH1 of the heavy chain or the corresponding region ofthe light chain.

The precise site at which the fusion is made is not critical; particularsites are well known and may be selected in order to optimize thebiological activity of the Bv8 and/or EG-VEGF immunoglobulin chimeras.

In some embodiments, the Bv8-immunoglobulin chimeras and/or EG-VEGFimmunoglobulin chimeras are assembled as monomers, or hetero- orhomo-multimer, and particularly as dimers or tetramers, essentially asillustrated in WO 91/08298.

In a preferred embodiment, the Bv8 and/or EG-VEGF sequence is fused tothe N-terminus of the C-terminal portion of an antibody (in particularthe Fc domain), containing the effector functions of an immunoglobulin,e.g. immunoglobulin G₁ (IgG1). It is possible to fuse the entire heavychain constant region to the Bv8 and/or EG-VEGF sequence. However, morepreferably, a sequence beginning in the hinge region just upstream ofthe papain cleavage site (which defines IgG Fc chemically; residue 216,taking the first residue of heavy chain constant region to be 114, oranalogous sites of other immunoglobulins) is used in the fusion. In aparticularly preferred embodiment, the Bv8 amino acid sequence and/orEG-VEGF amino acid sequence is fused to the hinge region and CH2 andCH3, or to the CH1, hinge, CH2 and CH3 domains of an IgG1, IgG2, or IgG3heavy chain. The precise site at which the fusion is made is notcritical, and the optimal site can be determined by routineexperimentation.

In some embodiments, the Bv8 and/or EG-VEGF immunoglobulin chimeras areassembled as multimer, and particularly as homo-dimers or -tetramers.Generally, these assembled immunoglobulins will have known unitstructures. A basic four chain structural unit is the form in which IgG,IgD, and IgE exist. A four-unit is repeated in the higher molecularweight immunoglobulins; IgM generally exists as a pentamer of basicfour-units held together by disulfide bonds. IgA globulin, andoccasionally IgG globulin, may also exist in multimeric form in serum.In the case of a multimer, each four-unit may be the same or different.

Alternatively, the Bv8 sequence or EG-VEGF sequence can be insertedbetween immunoglobulin heavy chain and light chain sequences such thatan immunoglobulin comprising a chimeric heavy chain is obtained. In thisembodiment, the Bv8 sequence or EG-VEGF sequence is fused to the 3′ endof an immunoglobulin heavy chain in each arm of an immunoglobulin,either between the hinge and the CH2 domain, or between the CH2 and CH3domains. Similar constructs have been reported by Hoogenboom et al.,Mol. Immunol., 28:1027-1037 (1991).

Although the presence of an immunoglobulin light chain is not requiredin the immunoadhesins of the present invention, an immunoglobulin lightchain might be present either covalently associated to a Bv8 or EG-VEGFimmunoglobulin heavy chain fusion polypeptide, or directly fused to Bv8or EG-VEGF. In the former case, DNA encoding an immunoglobulin lightchain is typically coexpressed with the DNA encoding the Bv8 or EG-VEGFimmunoglobulin heavy chain fusion protein. Upon secretion, the hybridheavy chain and the light chain will be covalently associated to providean immunoglobulin-like structure comprising two disulfide-linkedimmunoglobulin heavy chain-light chain pairs. Methods suitable for thepreparation of such structures are, for example, disclosed in U.S. Pat.No. 4,816,567 issued 28 Mar. 1989.

In a preferred embodiment, the immunoglobulin sequences used in theconstruction of the immunoadhesins of the present invention are from anIgG immunoglobulin heavy chain constant domain. For humanimmunoadhesins, the use of human IgG1 and IgG3 immunoglobulin sequencesis preferred. A major advantage of using IgG1 is that IgG1immunoadhesins can be purified efficiently on immobilized protein A. Incontrast, purification of IgG3 requires protein G, a significantly lessversatile medium. However, other structural and functional properties ofimmunoglobulins should be considered when choosing the Ig fusion partnerfor a particular immunoadhesin construction. For example, the IgG3 hingeis longer and more flexible, so it can accommodate larger adhesindomains that may not fold or function properly when fused to IgG1.Another consideration may be valency; IgG immunoadhesins are bivalenthomodimers, whereas Ig subtypes like IgA and IgM may give rise todimeric or pentameric structures, respectively, of the basic Ighomodimer unit. For Bv8 and/or EG-VEGF immunoadhesins designed for invivo application, the pharmacokinetic properties and the effectorfunctions specified by the Fc region are important as well. AlthoughIgG1, IgG2 and IgG4 all have in vivo half-lives of 21 days, theirrelative potencies at activating the complement system are different.IgG4 does not activate complement, and IgG2 is significantly weaker atcomplement activation than IgG1. Moreover, unlike IgG1, IgG2 does notbind to Fc receptors on mononuclear cells or neutrophils. While IgG3 isoptimal for complement activation, its in vivo half-life isapproximately one third of the other IgG isotypes. Another importantconsideration for immunoadhesins designed to be used as humantherapeutics is the number of allotypic variants of the particularisotype. In general, IgG isotypes with fewer serologically-definedallotypes are preferred. For example, IgG1 has only fourserologically-defined allotypic sites, two of which (G1m and 2) arelocated in the Fc region; and one of these sites G1m1, isnon-immunogenic. In contrast, there are 12 serologically-definedallotypes in IgG3, all of which are in the Fc region; only three ofthese sites (G3m5, 11 and 21) have one allotype which is nonimmunogenic.Thus, the potential immunogenicity of a γ3 immunoadhesin is greater thanthat of a γ1 immunoadhesin.

With respect to the parental immunoglobulin, a useful joining point isjust upstream of the cysteines of the hinge that form the disulfidebonds between the two heavy chains. In a frequently used design, thecodon for the C-terminal residue of the Bv8 or EG-VEGF part of themolecule is placed directly upstream of the codons for the sequenceDKTHTCPPCP of the IgG1 hinge region.

The general methods suitable for the construction and expression ofimmunoadhesins are the same as those disclosed hereinabove with regardto Bv8 and EG-VEGF. Bv8 immunoadhesins and EG-VEGF immunoadhesins aremost conveniently constructed by fusing the cDNA sequence encoding theBv8 portion or EG-VEGF portion respectively in-frame to an Ig cDNAsequence. However, fusion to genomic Ig fragments can also be used (see,e.g., Gascoigne et al., 1987, Proc. Natl. Acad. Sci. USA, 84:2936-2940;Aruffo et al., 1990, Cell, 61:1303-1313; Stamenkovic et al., 1991, Cell,66:1133-1144). The latter type of fusion requires the presence of Igregulatory sequences for expression. cDNAs encoding IgG heavy-chainconstant regions can be isolated based on published sequence from cDNAlibraries derived from spleen or peripheral blood lymphocytes, byhybridization or by polymerase chain reaction (PCR) techniques. ThecDNAs encoding the Bv8 or EG-VEGF and Ig parts of the immunoadhesin areinserted in tandem into a plasmid vector that directs efficientexpression in the chosen host cells. For expression in mammalian cells,pRK5-based vectors (Schall et al., 1990, Cell, 61:361-370) andCDM8-based vectors (Seed, 1989, Nature, 329:840) can be used. The exactjunction can be created by removing the extra sequences between thedesigned junction codons using oligonucleotide-directed deletionalmutagenesis (Zoller et al., 1982, Nucleic Acids Res., 10:6487; Capon etal., 1989, Nature, 337:525-531). Synthetic oligonucleotides can be used,in which each half is complementary to the sequence on either side ofthe desired junction; ideally, these are 36 to 48-mers. Alternatively,PCR techniques can be used to join the two parts of the moleculein-frame with an appropriate vector.

The choice of host cell line for the expression of Bv8 or EG-VEGFimmunoadhesins depends mainly on the expression vector. Anotherconsideration is the amount of protein that is required. Milligramquantities often can be produced by transient transfections. Forexample, the adenovirus EIA-transformed 293 human embryonic kidney cellline can be transfected transiently with pRK5-based vectors by amodification of the calcium phosphate method to allow efficientimmunoadhesin expression. CDM8-based vectors can be used to transfectCOS cells by the DEAE-dextran method (Aruffo et al., 1990, Cell,61:1303-1313; Zettmeissl et al., 1990, DNA Cell Biol. US, 9:347-353). Iflarger amounts of protein are desired, the immunoadhesin can beexpressed after stable transfection of a host cell line. For example, apRK5-based vector can be introduced into Chinese hamster ovary (CHO)cells in the presence of an additional plasmid encoding dihydrofolatereductase (DHFR) and conferring resistance to G418. Clones resistant toG418 can be selected in culture; these clones are grown in the presenceof increasing levels of DHFR inhibitor methotrexate; clones areselected, in which the number of gene copies encoding the DHFR andimmunoadhesin sequences is co-amplified. If the immunoadhesin contains ahydrophobic leader sequence at its N-terminus, it is likely to beprocessed and secreted by the transfected cells. The expression ofimmunoadhesins with more complex structures may require uniquely suitedhost cells; for example, components such as light chain or J chain maybe provided by certain myeloma or hybridoma cell hosts (Gascoigne etal., 1987, supra, Martin et al., 1993, J. Virol., 67:3561-3568).

Immunoadhesins can be conveniently purified by affinity chromatography.The suitability of protein A as an affinity ligand depends on thespecies and isotype of the immunoglobulin Fc domain that is used in thechimera. Protein A can be used to purify immunoadhesins that are basedon human γ1, γ2, or γ4 heavy chains (Lindmark et al., J. Immunol. Meth.,62:1-13 (1983)). Protein G is recommended for all mouse isotypes and forhuman γ3 (Guss et al., 1986, EMBO J., 5:1567-1575). The matrix to whichthe affinity ligand is attached is most often agarose, but othermatrices are available. Mechanically stable matrices such as controlledpore glass or poly(styrenedivinyl)benzene allow for faster flow ratesand shorter processing times than can be achieved with agarose. Theconditions for binding an immunoadhesin to the protein A or G affinitycolumn are dictated entirely by the characteristics of the Fc domain;that is, its species and isotype. Generally, when the proper ligand ischosen, efficient binding occurs directly from unconditioned culturefluid. One distinguishing feature of immunoadhesins is that, for humanγ1 molecules, the binding capacity for protein A is somewhat diminishedrelative to an antibody of the same Fc type. Bound immunoadhesin can beefficiently eluted either at acidic pH (at or above 3.0), or in aneutral pH buffer containing a mildly chaotropic salt. This affinitychromatography step can result in an immunoadhesin preparation thatis >95% pure.

Other methods known in the art can be used in place of, or in additionto, affinity chromatography on protein A or G to purify immunoadhesins.Immunoadhesins behave similarly to antibodies in thiophilic gelchromatography (Hutchens et al., 1986, Anal. Biochem., 159:217-226) andimmobilized metal chelate chromatography (Al-Mashikhi et al., 1988, J.Dairy Sci., 71:1756-1763). In contrast to antibodies, however, theirbehavior on ion exchange columns is dictated not only by theirisoelectric points, but also by a charge dipole that may exist in themolecules due to their chimeric nature.

If desired, the immunoadhesins can be made bispecific. Thus, theimmunoadhesins of the present invention may combine a Bv8 or EG-VEGFdomain and a domain, such as a domain from another growth factorincluding, but not limited to VEGF, Bv8, and EG-VEGF. For bispecificmolecules, trimeric molecules, composed of a chimeric antibody heavychain in one arm and a chimeric antibody heavy chain-light chain pair inthe other arm of their antibody-like structure are advantageous, due toease of purification. In contrast to antibody-producing quadromastraditionally used for the production of bispecific immunoadhesins,which produce a mixture of ten tetramers, cells transfected with nucleicacid encoding the three chains of a trimeric immunoadhesin structureproduce a mixture of only three molecules, and purification of thedesired product from this mixture is correspondingly easier.

F. Modulators of Bv8 Activity and EG-VEGF Activity

The present invention also encompasses methods of screening compounds toidentify those that mimic or enhance one or more biological activity ofBv8 and/or EG-VEGF (agonists) or prevent the effect of Bv8 and/orEG-VEGF (antagonists). Bv8 and EG-VEGF agonists and antagonists are alsoreferred to as Bv8 and EG-VEGF modulators, respectively. Screeningassays for antagonist drug candidates are designed to identify compoundsthat bind or complex with Bv8 polypeptides and/or EG-VEGF polypeptide,or otherwise interfere with the interaction of Bv8 and/or EG-VEGF withother cellular proteins.

1. Small Molecule Screening

Small molecules may have the ability to act as Bv8 agonists orantagonists and/or EG-VEGF agonists or antagonists and thus to betherapeutically useful. Such small molecules may include naturallyoccurring small molecules, synthetic, organic, or inorganic compoundsand peptides. However, small molecules in the present invention are notlimited to these forms. Extensive libraries of small molecules arecommercially available and a wide variety of assays are well known inthe art to screen these molecules for the desired activity.

Candidate Bv8 agonist or antagonist and/or EG-VEGF agonist or antagonistsmall molecules are preferably identified first in an assay that allowsfor the rapid identification of potential modulators of Bv8 and/orEG-VEGF activity. An example of such an assay is a protein-proteinbinding assay wherein the ability of the candidate molecule to bind to aBv8 or EG-VEGF receptor is measured. In another example, the ability ofcandidate molecules to interfere with Bv8 binding to a Bv8 receptor orEG-VEGF binding to an EG-VEGF receptor is measured. In an embodiment,the respective Bv8 receptor or EG-VEGF receptor is Bv8/EG-VEGFreceptor-1 and/or Bv8/EG-VEGF receptor-2.

In a preferred embodiment, small molecule Bv8 agonists and EG-VEGF areidentified by their ability to mimic one or more of the biologicalactivities of Bv8 or EG-VEGF, respectively. For example, candidatecompounds are screened for their ability to induce proliferation ofendothelial cells, to promote endothelial cell survival, or to induceangiogenesis, as described below. Candidate compounds that induce one ormore of the described biological activities of Bv8 or EG-VEGF areidentified as agonists.

In another embodiment, small molecule Bv8 antagonists and/or EG-VEGFantagonists are identified by their ability to inhibit one or more ofthe biological activities of Bv8 and/or EG-VEGF, respectively. Forexample, candidate compounds are screened for their ability to inhibitproliferation of endothelial cells, endothelial cell survival, orangiogenesis, as described below. Candidate compounds that inhibit oneor more of the described biological activities of Bv8 or EG-VEGF areidentified as antagonists.

The ability of a candidate compound to induce or inhibit angiogenesis isdetermined, for example, in mice testes as described in WO 02/00711 andWO 03/020892.

Endothelial cell proliferation is determined, for example, as describedin WO 02/00711 and WO 03/020892. Briefly, endothelial cells are grown inlow glucose DMEM supplemented with 10% bovine serum albumin containing acandidate agonist compound or Bv8 and/or EG-VEGF that has been contactedwith a candidate antagonist compound. The endothelial cells are platedat a density of 4000 to 6000 cells/ml in 6 or 12 well dishes. At day 5to 7 of the assay, the endothelial cells are trypsinized and the numberof cells is quantitated using a Coulter counter.

Endothelial cell survival is determined, for example, as described in WO02/00711 and WO 03/020892. Briefly, approximately 2×10⁵ bovine braincapillary cells are plated in low glucose DMEM supplemented with 10%bovine serum albumin and incubated for 24 hours. The media is thenaspirated and replaced with media containing a candidate agonistcompound or Bv8 and/or EG-VEGF that has been contacted with a candidateantagonist compound. The cells are incubated for 48 hours, trypsinized,and fixed in 70% ethanol. The fixed cells are stained with propidiumiodine and RNase and the sub-G1 profile of the cells is determined byFACs analysis.

Compounds identified as Bv8 and/or EG-VEGF agonists or antagonists maybe used in the methods of the present invention. For example, Bv8 and/orEG-VEGF antagonists may be used to treat cancer.

2. Preparation and Identification of Antibodies

Agonist human and non-human polyclonal and monoclonal antibodies(including humanized forms of non-human monoclonal antibodies), whichmimic the biological properties of Bv8 and/or EG-VEGF, and antagonisthuman and non-human polyclonal and monoclonal antibodies (includinghumanized forms of non-human monoclonal antibodies), which inhibit thebiological properties of Bv8 and/or EG-VEGF, are also contemplated inthe present invention. These include amino acid sequence variants,glycosylation variants and fragments of antibodies. General techniquesfor the production of such antibodies and the selection of agonistantibodies are known in the art and are briefly described below.

(i) Polyclonal Antibodies

Methods of preparing polyclonal antibodies are known in the art.Polyclonal antibodies can be raised in a mammal, for example, by one ormore injections of an immunizing agent and, if desired, an adjuvant.Typically, the immunizing agent and/or adjuvant will be injected in themammal by multiple subcutaneous or intraperitoneal injections. It may beuseful to conjugate the immunizing agent to a protein known to beimmunogenic in the mammal being immunized, such as serum albumin, orsoybean trypsin inhibitor. Examples of adjuvants that may be employedinclude Freund's complete adjuvant and MPL-TDM.

(ii) Monoclonal Antibodies

Monoclonal antibodies may be made using the hybridoma method firstdescribed by Kohler et al., 1975, Nature, 256:495, or may be made byrecombinant DNA methods (U.S. Pat. No. 4,816,567).

In the hybridoma method, a mouse or other appropriate host animal, suchas a hamster or macaque monkey, is immunized as hereinabove described toelicit lymphocytes that produce or are capable of producing antibodiesthat will specifically bind to the protein used for immunization.Alternatively, lymphocytes may be immunized in vitro. Lymphocytes thenare fused with myeloma cells using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell (Goding, MonoclonalAntibodies: Principles and Practice, pp. 59-103, (Academic Press,1986)).

The hybridoma cells thus prepared are seeded and grown in a suitableculture medium that preferably contains one or more substances thatinhibit the growth or survival of the unfused, parental myeloma cells.For example, if the parental myeloma cells lack the enzyme hypoxanthineguanine phosphoribosyl transferase (HGPRT or HPRT), the culture mediumfor the hybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), conditions under which the growth ofHGPRT-deficient cells is prevented.

Preferred myeloma cells are those that fuse efficiently, support stablehigh-level production of antibody by the selected antibody-producingcells, and are sensitive to a medium such as HAT medium. Among these,preferred myeloma cell lines are murine myeloma lines, such as thosederived from MOP-21 and M.C.-11 mouse tumors available from the SalkInstitute Cell Distribution Center, San Diego, Calif. USA, and SP-2 orX63-Ag8-653 cells available from the American Type Culture Collection,Rockville, Md. USA. Human myeloma and mouse-human heteromyeloma celllines also have been described for the production of human monoclonalantibodies (Kozbor, 1984, J. Immunol., 133:3001; Brodeur et al.,Monoclonal Antibody Production Techniques and Applications, pp. 51-63,Marcel Dekker, Inc., New York, (1987)).

Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against the antigen.Preferably, the binding specificity of monoclonal antibodies produced byhybridoma cells is determined by immunoprecipitation or by an in vitrobinding assay, such as radioimmunoassay (RIA) or enzyme-linkedimmunoabsorbent assay (ELISA).

The binding affinity of the monoclonal antibody can, for example, bedetermined by the Scatchard analysis of Munson et al., 1980, Anal.Biochem., 107:220.

After hybridoma cells are identified that produce antibodies of thedesired specificity, affinity, and/or activity, the cells may besubcloned by limiting dilution procedures and grown by standard methods(Goding, Monoclonal Antibodies: Principles and Practice, pp.59-103(Academic Press, 1986)). Suitable culture media for this purposeinclude, for example, DMEM or RPMI-1640 medium. In addition, thehybridoma cells may be grown in vivo as ascites tumors in an animal.

The monoclonal antibodies secreted by the subclones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

DNA encoding the monoclonal antibodies is readily isolated and sequencedusing conventional procedures (e.g., by using oligonucleotide probesthat are capable of binding specifically to genes encoding the heavy andlight chains of the monoclonal antibodies). The hybridoma cells serve asa preferred source of such DNA. Once isolated, the DNA may be placedinto expression vectors, which are then transfected into host cells suchas E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells,or myeloma cells that do not otherwise produce immunoglobulin protein,to obtain the synthesis of monoclonal antibodies in the recombinant hostcells. The DNA also may be modified, for example, by substituting thecoding sequence for human heavy and light chain constant domains inplace of the homologous murine sequences, Morrison, et al., 1984, Proc.Nat. Acad. Sci. U.S.A., 81:6851, or by covalently joining to theimmunoglobulin coding sequence all or part of the coding sequence for anon-immunoglobulin polypeptide. In that manner, “chimeric” or “hybrid”antibodies are prepared that have the binding specificity of a Bv8 orEG-VEGF agonist monoclonal antibody or Bv8 or EG-VEGF antagonistmonoclonal antibody described herein.

Chimeric or hybrid antibodies also may be prepared in vitro using knownmethods in synthetic protein chemistry, including those involvingcrosslinking agents. For example, immunotoxins may be constructed usinga disulfide exchange reaction or by forming a thioether bond. Examplesof suitable reagents for this purpose include iminothiolate andmethyl-4-mercaptobutyrimidate.

Recombinant production of antibodies will be described in more detailbelow.

(iii) Humanized Antibodies

Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a non-human source. These non-human amino acidresidues are often referred to as “import” residues, which are typicallytaken from an “import” variable domain. Humanization can be essentiallyperformed following the method of Winter and co-workers (Jones et al.,1986, Nature, 321:522-525; Riechmann et al., 1988, Nature, 332:323-327;Verhoeyen et al., 1988, Science, 239:1534-1536), by substituting rodentCDRs or CDR sequences for the corresponding sequences of a humanantibody.

Accordingly, such “humanized” antibodies are chimeric antibodies(Cabilly, supra), wherein substantially less than an intact humanvariable domain has been substituted by the corresponding sequence froma non-human species. In practice, humanized antibodies are typicallyhuman antibodies in which some CDR residues and possibly some FRresidues are substituted by residues from analogous sites in rodentantibodies.

It is important that antibodies be humanized with retention of highaffinity for the antigen and other favorable biological properties. Toachieve this goal, according to a preferred method, humanized antibodiesare prepared by a process of analysis of the parental sequences andvarious conceptual humanized products using three-dimensional models ofthe parental and humanized sequences. Three dimensional immunoglobulinmodels are commonly available and are familiar to those skilled in theart. Computer programs are available which illustrate and displayprobable three-dimensional conformational structures of selectedcandidate immunoglobulin sequences. Inspection of these displays permitsanalysis of the likely role of the residues in the functioning of thecandidate immunoglobulin sequence, i.e. the analysis of residues thatinfluence the ability of the candidate immunoglobulin to bind itsantigen. In this way, FR residues can be selected and combined from theconsensus and import sequence so that the desired antibodycharacteristic, such as increased affinity for the target antigen(s), isachieved. In general, the CDR residues are directly and mostsubstantially involved in influencing antigen binding. For furtherdetails see U.S. Pat. Nos. 5,821,337 and 6,054,297.

(iv) Human Antibodies

Human monoclonal antibodies can be made by the hybridoma method. Humanmyeloma and mouse-human heteromyeloma cell lines for the production ofhuman monoclonal antibodies have been described, for example, by Kozbor,1984, J. Immunol. 133, 3001, and Brodeur, et al., Monoclonal AntibodyProduction Techniques and Applications, pages 51-63 (Marcel Dekker,Inc., New York, 1987).

It is now possible to produce transgenic animals (e.g., mice) that arecapable, upon immunization, of producing a repertoire of humanantibodies in the absence of endogenous immunoglobulin production. Forexample, it has been described that the homozygous deletion of theantibody heavy chain joining region (J_(H)) gene in chimeric andgerm-line mutant mice results in complete inhibition of endogenousantibody production. Transfer of the human germ-line immunoglobulin genearray in such germ-line mutant mice will result in the production ofhuman antibodies upon antigen challenge. See, e.g. Jakobovits et al.,1993, Proc. Natl. Acad. Sci. USA 90, 2551-255; Jakobovits et al., 1993,Nature 362, 255-258.

Mendez et al. (1997, Nature Genetics 15:146-156) have further improvedthe technology and have generated a line of transgenic mice designatedas “Xenomouse II” that, when challenged with an antigen, generates highaffinity fully human antibodies. This was achieved by germ-lineintegration of megabase human heavy chain and light chain loci into micewith deletion into endogenous J_(H) segment as described above. TheXenomouse II harbors 1,020 kb of human heavy chain locus containingapproximately 66 V_(H) genes, complete D_(H) and J_(H) regions and threedifferent constant regions (μ, δ and χ), and also harbors 800 kb ofhuman κ locus containing 32 Vκ genes, Jκ segments and Cκ genes. Theantibodies produced in these mice closely resemble that seen in humansin all respects, including gene rearrangement, assembly, and repertoire.The human antibodies are preferentially expressed over endogenousantibodies due to deletion in endogenous J_(H) segment that preventsgene rearrangement in the murine locus.

Alternatively, phage display technology (McCafferty et al., 1990, Nature348:552-553) can be used to produce human antibodies and antibodyfragments in vitro, from immunoglobulin variable (V) domain generepertoires from unimmunized donors. According to this technique,antibody V domain genes are cloned in-frame into either a major or minorcoat protein gene of a filamentous bacteriophage, such as M13 or fd, anddisplayed as functional antibody fragments on the surface of the phageparticle. Because the filamentous particle contains a single-strandedDNA copy of the phage genome, selections based on the functionalproperties of the antibody also result in selection of the gene encodingthe antibody exhibiting those properties. Thus, the phage mimics some ofthe properties of the B-cell. Phage display can be performed in avariety of formats; for their review see, e.g. Johnson, Kevin S. andChiswell, David J., 1993, Current Opinion in Structural Biology3:564-571. Several sources of V-gene segments can be used for phagedisplay. Clackson et al., 1991, Nature 352:624-628 isolated a diversearray of anti-oxazolone antibodies from a small random combinatoriallibrary of V genes derived from the spleens of immunized mice. Arepertoire of V genes from unimmunized human donors can be constructedand antibodies to a diverse array of antigens (including self-antigens)can be isolated essentially following the techniques described by Markset al., 1991, J. Mol. Biol. 222:581-597, or Griffith et al., 1993, EMBOJ. 12:725-734. In a natural immune response, antibody genes accumulatemutations at a high rate (somatic hypermutation). Some of the changesintroduced will confer higher affinity, and B cells displayinghigh-affinity surface immunoglobulin are preferentially replicated anddifferentiated during subsequent antigen challenge. This natural processcan be mimicked by employing the technique known as “chain shuffling”(Marks et al., 1992, BioTechnol., 10:779-783). In this method, theaffinity of “primary” human antibodies obtained by phage display can beimproved by sequentially replacing the heavy and light chain V regiongenes with repertoires of naturally occurring variants (repertoires) ofV domain genes obtained from unimmunized donors. This technique allowsthe production of antibodies and antibody fragments with affinities inthe nM range. A strategy for making very large phage antibodyrepertoires (also known as “the mother-of-all libraries”) has beendescribed by Waterhouse et al., 1993, Nucl. Acids Res. 21:2265-2266, andthe isolation of a high affinity human antibody directly from such largephage library is reported by Griffith et al., 1993, EMBO J. 12:725-734.Gene shuffling can also be used to derive human antibodies from rodentantibodies, where the human antibody has similar affinities andspecificities to the starting rodent antibody. According to this method,which is also referred to as “epitope imprinting”, the heavy or lightchain V domain gene of rodent antibodies obtained by phage displaytechnique is replaced with a repertoire of human V domain genes,creating rodent-human chimeras. Selection on antigen results inisolation of human variable domains capable of restoring a functionalantigen-binding site, i.e. the epitope governs (imprints) the choice ofpartner. When the process is repeated in order to replace the remainingrodent V domain, a human antibody is obtained (see PCT patentapplication WO 93/06213, published 1 Apr. 1993). Unlike traditionalhumanization of rodent antibodies by CDR grafting, this techniqueprovides completely human antibodies, which have no framework or CDRresidues of rodent origin.

(v) Bispecific Antibodies

Bispecific antibodies are monoclonal, preferably human or humanized,antibodies that have binding specificities for at least two differentantigens. Methods for making bispecific antibodies are known in the art.Traditionally, the recombinant production of bispecific antibodies isbased on the coexpression of two immunoglobulin heavy chain-light chainpairs, where the two heavy chains have different specificities(Millstein and Cuello, Nature 305, 537-539 (1983)). Because of therandom assortment of immunoglobulin heavy and light chains, thesehybridomas (quadromas) produce a potential mixture of 10 differentantibody molecules, of which only one has the correct bispecificstructure. The purification of the correct molecule, which is usuallydone by affinity chromatography steps, is rather cumbersome, and theproduct yields are low. Similar procedures are disclosed in PCTapplication publication No. WO 93/08829 (published 13 May 1993), and inTraunecker et al., 1991, EMBO 10, 3655-3659.

According to a different and more preferred approach, antibody variabledomains with the desired binding specificities (antibody-antigencombining sites) are fused to immunoglobulin constant domain sequences.The fusion preferably is with an immunoglobulin heavy chain constantdomain, comprising at least part of the hinge, CH2 and CH3 regions. Itis preferred to have the first heavy chain constant region (CH1)containing the site necessary for light chain binding, present in atleast one of the fusions. DNAs encoding the immunoglobulin heavy chainfusions and, if desired, the immunoglobulin light chain, are insertedinto separate expression vectors, and are cotransfected into a suitablehost organism. This provides for great flexibility in adjusting themutual proportions of the three polypeptide fragments in embodimentswhen unequal ratios of the three polypeptide chains used in theconstruction provide the optimum yields. It is, however, possible toinsert the coding sequences for two or all three polypeptide chains inone expression vector when the expression of at least two polypeptidechains in equal ratios results in high yields or when the ratios are ofno particular significance. In a preferred embodiment of this approach,the bispecific antibodies are composed of a hybrid immunoglobulin heavychain with a first binding specificity in one arm, and a hybridimmunoglobulin heavy chain-light chain pair (providing a second bindingspecificity) in the other arm. It was found that this asymmetricstructure facilitates the separation of the desired bispecific compoundfrom unwanted immunoglobulin chain combinations, as the presence of animmunoglobulin light chain in only one half of the bispecific moleculeprovides for a facile way of separation. This approach is disclosed inPCT Publication No. WO 94/04690, published on Mar. 3, 1994.

For further details of generating bispecific antibodies see, forexample, Suresh et al., 1986, Methods in Enzymology 121, 210.

(vi) Heteroconjugate Antibodies

Heteroconjugate antibodies are composed of two covalently joinedantibodies. Such antibodies have, for example, been proposed to targetimmune system cells to unwanted cells (U.S. Pat. No. 4,676,980), and fortreatment of HIV infection (PCT application publication Nos. WO 91/00360and WO 92/200373; EP 03089). Heteroconjugate antibodies may be madeusing any convenient cross-linking methods. Suitable cross-linkingagents are well known in the art, and are disclosed in U.S. Pat. No.4,676,980, along with a number of cross-linking techniques.

(vii) Antibody Fragments

In certain embodiments, the agonist and/or antagonist antibody(including murine, human and humanized antibodies and antibody variants)is an antibody fragment. Various techniques have been developed for theproduction of antibody fragments. Traditionally, these fragments werederived via proteolytic digestion of intact antibodies (see, e.g.,Morimoto et al., 1992, J. Biochem. Biophys. Methods 24:107-117 andBrennan et al., 1985, Science 229:81). However, these fragments can nowbe produced directly by recombinant host cells. For example, Fab′-SHfragments can be directly recovered from E. coli and chemically coupledto form F(ab′)2 fragments (Carter et al., 1992, Bio/Technology10:163-167). In another embodiment, the F(ab′)₂ is formed using theleucine zipper GCN4 to promote assembly of the F(ab′)₂ molecule.According to another approach, Fv, Fab or F(ab′)₂ fragments can beisolated directly from recombinant host cell culture. Other techniquesfor the production of antibody fragments will be apparent to the skilledpractitioner.

(viii) Identification of Agonist Antibodies

Bv8 agonist antibodies and EG-VEGF agonist antibodies are identifiedbased on their ability to induce a biological activity of Bv8 orEG-VEGF, including but not limited to proliferation of endothelialcells, survival of endothelial cells, and angiogenesis. Assays fordetermining proliferation of endothelial cells, survival of endothelialcells, and angiogenesis are known in the art. Induction of proliferationof endothelial cells can be assayed as described above under smallmolecules. Bv8 agonist antibodies and EG-VEGF agonist antibodies can beidentified by their ability to induce angiogenesis, for example in micetestes, as described in WO 02/00711 and WO 03/020892.

(ix) Identification of Antagonist Antibodies

Bv8 antagonist antibodies and EG-VEGF antagonist antibodies areidentified based on their ability to inhibit a biological activity ofBv8 or EG-VEGF, including but not limited to proliferation ofendothelial cells, survival of endothelial cells, and angiogenesis, forexample, using one of the assays described above.

G. Screening for Proteins that Interact with Bv8 and/or EG-VEGF

Any method suitable for detecting protein-protein interactions may beemployed for identifying proteins or other molecules, including but notlimited to transmembrane or intracellular proteins, that interact withBv8 and/or EG-VEGF. Among the traditional methods that may be employedare co-immunoprecipitation, crosslinking and co-purification throughgradients or chromatographic columns to identify proteins that interactwith Bv8 or EG-VEGF. For such assays, the Bv8 component or EG-VEGFcomponent can be a full-length protein, a soluble derivative thereof, apeptide corresponding to a domain of interest, or a fusion proteincontaining some region of Bv8 or EG-VEGF.

Methods may be employed which result in the simultaneous identificationof genes that encode proteins capable of interacting with Bv8 orEG-VEGF. These methods include, for example, probing expressionlibraries, in a manner similar to the well-known technique of antibodyprobing of λgt11 libraries, using labeled Bv8, labeled EG-VEGF, or alabeled variant thereof.

A method that detects protein interactions in vivo, the two-hybridsystem, is described in detail for illustration only and not by way oflimitation. One version of this system has been described (Chien et al.,1991, Proc. Natl. Acad. Sci. USA, 88:9578-9582) and is commerciallyavailable from Clontech (Palo Alto, Calif.).

Briefly, utilizing such a system, plasmids are constructed that encodetwo hybrid proteins: one plasmid consists of nucleotides encoding theDNA-binding domain of a transcription activator protein fused to anucleotide sequence encoding Bv8 or EG-VEGF, or a polypeptide, peptide,or fusion protein thereof, and the other plasmid consists of nucleotidesencoding the transcription activator protein's activation domain fusedto a cDNA encoding an unknown protein which has been recombined intothis plasmid as part of a cDNA library. The DNA-binding domain fusionplasmid and the cDNA library are transformed into a strain of the yeastSaccharomyces cerevisiae that contains a reporter gene (e.g. HBS orlacZ) whose regulatory region contains the transcription activator'sbinding site. Either hybrid protein alone cannot activate transcriptionof the reporter gene: the DNA-binding domain hybrid cannot because itdoes not provide activation function and the activation domain hybridcannot because it cannot localize to the activator's binding sites.Interaction of the two hybrid proteins reconstitutes the functionalactivator protein and results in expression of the reporter gene, whichis detected by an assay for the reporter gene product.

The two-hybrid system or related methodology may be used to screenactivation domain libraries for proteins that interact with the “bait”gene product. By way of example, and not by way of limitation, Bv8 orEG-VEGF can be used as the bait gene product. Total genomic or cDNAsequences are fused to the DNA encoding an activation domain. Thislibrary and a plasmid encoding a hybrid of a bait Bv8 gene product orbait EG-VEGF gene product fused to the DNA-binding domain arecotransformed into a yeast reporter strain, and the resultingtransformants are screened for those that express the reporter gene. Forexample, and not by way of limitation, a bait Bv8 gene sequence or baitEG-VEGF gene sequence, for example, the genes open reading frame of thegene, can be cloned into a vector such that it is translationally fusedto the DNA encoding the DNA-binding domain of the GAL4 protein. Thesecolonies are purified and the library plasmids responsible for reportergene expression are isolated. DNA sequencing is then used to identifythe proteins encoded by the library plasmids.

A cDNA library of the cell line from which proteins that interact withthe bait Bv8 gene product or bait EG-VEGF gene product are to bedetected can be made using methods routinely practiced in the art.According to the particular system described herein, for example, thecDNA fragments can be inserted into a vector such that they aretranslationally fused to the transcriptional activation domain of GAL4.This library can be co-transformed along with the bait Bv8 gene-GAL4fusion plasmid or bait EG-VEGF gene-GAL4 fusion plasmid into a yeaststrain that contains a lacZ gene driven by a promoter which contains aGAL4 activation sequence. A cDNA encoded protein, fused to GAL4transcriptional activation domain, that interacts with the bait Bv8 geneproduct or bait EG-VEGF gene product will reconstitute an active GAL4protein and thereby drive expression. Colonies that drive expression canbe detected by methods routine in the art. The cDNA can then be purifiedfrom these strains, and used to produce and isolate the bait Bv8gene-interacting protein or bait EG-VEGF gene-interacting protein usingtechniques routinely practiced in the art.

1. Compounds that Modulate Bv8 and/or EG-VEGF Expression or Activity

The following assays are designed to identify compounds that interactwith (e.g., bind to) Bv8 or EG-VEGF compounds that interfere with theinteraction of Bv8 or EG-VEGF with their binding partners, cognatereceptor, and to compounds that modulate the activity of Bv8 geneexpression and/or EG-VEGF gene expression (that is, modulate the levelof Bv8 gene expression and/or EG-VEGF gene expression) or modulate thelevels of Bv8 and/or EG-VEGF in the body. Assays can also be used toidentify compounds that bind Bv8 and/or EG-VEGF gene regulatorysequences (for example, promoter sequences) and, consequently, modulateBv8 and/or EG-VEGF gene expression. See, for example, Platt, K. A.,1994, J. Biol. Chem. 269:28558-28562, which is incorporated herein byreference in its entirety.

Compounds that may be screened in accordance with the invention include,but are not limited to peptides, soluble receptors or fragments thereof,antibodies and fragments thereof, and other organic compounds (e.g.,peptidomimetics) that bind to Bv8 or EG-VEGF, or to a Bv8/EG-VEGFreceptor and either mimic the activity triggered by a natural ligand(agonists) or inhibit the activity triggered by the natural ligand(antagonists).

Such compounds may include, but are not limited to, peptides such as,for example, soluble peptides, including but not limited to members ofrandom peptide libraries; (see, for example, Lam, K. S. et al., 1991,Nature 354:82-84; Houghten, R. et al., 1991, Nature 354:84-86), andcombinatorial chemistry-derived molecular library made of D- and/orL-configuration amino acids, phosphopeptides (including, but not limitedto members of random or partially degenerate, directed phosphopeptidelibraries; see, for example, Songyang, Z. et al., 1993, Cell72:767-778), antibodies (including, but not limited to, polyclonal,monoclonal, humanized, anti-idiotypic, chimeric, or single chainantibodies, and FAb, F(ab′)₂, and FAb expression library fragments, andepitope-binding fragments thereof), and small organic or inorganicmolecules.

Other compounds that can be screened in accordance with the inventioninclude, but are not limited to small organic molecules that are able togain entry into an appropriate cell (for example, an endothelial cell)and affect the expression of a Bv8 gene or an EG-VEGF gene, or someother gene involved in a Bv8 and/or EG-VEGF mediated pathway (e.g., byinteracting with the regulatory region or transcription factors involvedin gene expression); or such compounds that affect or substitute for theactivity of Bv8 or EG-VEGF or the activity of some other intracellularfactor involved in a Bv8 and/or EG-VEGF signal transduction, catabolic,or metabolic pathways.

Computer modeling and searching technologies permit identification ofcompounds, or the improvement of already identified compounds, that canmodulate Bv8 or EG-VEGF expression or activity. Having identified such acompound or composition, the active sites or regions are identified.Such active sites might typically be ligand binding sites. The activesite can be identified using methods known in the art including, forexample, from the amino acid sequences of peptides, from the nucleotidesequences of nucleic acids, or from study of complexes of the relevantcompound or composition with its natural ligand. In the latter case,chemical or X-ray crystallographic methods can be used to find theactive site by finding where on the factor the complexed ligand isfound.

Next, the three dimensional geometric structure of the active site isdetermined. This can be done by known methods, including X-raycrystallography, that can determine a complete molecular structure. Onthe other hand, solid or liquid phase NMR can be used to determinecertain intra-molecular distances. Any other experimental method ofstructure determination can be used to obtain partial or completegeometric structures. The geometric structures may be measured with acomplexed ligand, natural or artificial, which may increase the accuracyof the active site structure determined.

If an incomplete or insufficiently accurate structure is determined, themethods of computer based numerical modeling can be used to complete thestructure or improve its accuracy. Any recognized modeling method may beused, including parameterized models specific to particular biopolymerssuch as proteins or nucleic acids, molecular dynamics models based oncomputing molecular motions, statistical mechanics models based onthermal ensembles, or combined models. For most types of models,standard molecular force fields, representing the forces betweenconstituent atoms and groups, are necessary, and can be selected fromforce fields known in physical chemistry. The incomplete or lessaccurate experimental structures can serve as constraints on thecomplete and more accurate structures computed by these modelingmethods.

Finally, having determined the structure of the active site (or bindingsite), either experimentally, by modeling, or by a combination,candidate modulating compounds can be identified by searching databasescontaining compounds along with information on their molecularstructure. Such a search seeks compounds having structures that matchthe determined active site structure and that interact with the groupsdefining the active site. Such a search can be manual, but is preferablycomputer assisted. These compounds found from this search are potentialmodulators of Bv8 activity and/or EG-VEGF activity.

Alternatively, these methods can be used to identify improved modulatingcompounds from an already known modulating compound or ligand. Thecomposition of the known compound can be modified and the structuraleffects of modification can be determined using the experimental andcomputer modeling methods described above applied to the newcomposition. The altered structure is then compared to the active sitestructure of the compound to determine if an improved fit or interactionresults. In this manner systematic variations in composition, such as byvarying side groups, can be quickly evaluated to obtain modifiedmodulating compounds or ligands of improved specificity or activity.

Further experimental and computer modeling methods useful to identifymodulating compounds based upon identification of the active sites (orbinding sites) of Bv8 or EG-VEGF, and related transduction andtranscription factors will be apparent to those of skill in the art.

Examples of molecular modeling systems are the CHARMm and QUANTAprograms (Polygen Corporation, Waltham, Mass.). CHARMm performs theenergy minimization and molecular dynamics functions. QUANTA performsthe construction, graphic modeling and analysis of molecular structure.QUANTA allows interactive construction, modification, visualization, andanalysis of the behavior of molecules with each other.

A number of articles review computer modeling of drugs interactive withspecific proteins, such as Rotivinen, et al., 1988, Acta PharmaceuticalFennica 97:159-166; Ripka, Jun. 16, 1988, New Scientist 54-57; McKinalyand Rossmann, 1989, Annu. Rev. Pharmacol Toxiciol. 29:111-122; Perry andDavies, OSAR: Quantitative Structure-Activity Relationships in DrugDesign pp. 189-193 (Alan R. Liss, Inc. 1989); Lewis and Dean, 1989 Proc.R. Soc. Lond. 236:125-140 and 141-162; and, with respect to a modelreceptor for nucleic acid components, Askew, et al., 1989, J Am. Chem.Soc. 111:1082-1090. Other computer programs that screen and graphicallydepict chemicals are available from companies such as BioDesign, Inc.(Pasadena, Calif.), Allelix, Inc. (Mississauga, Ontario, Canada), andHypercube, Inc. (Cambridge, Ontario). Although these are primarilydesigned for application to drugs specific to particular proteins, theycan be adapted to design of drugs specific to regions of DNA or RNA,once that region is identified.

Although described above with reference to design and generation ofcompounds which could alter binding, one could also screen libraries ofknown compounds, including natural products or synthetic chemicals, andbiologically active materials, including proteins, for compounds whichare inhibitors or activators.

Compounds identified via assays such as those described herein may beuseful, for example, in elucidating the biological function of a Bv8gene product and/or a EG-VEGF gene product. Such compounds can beadministered to a patient at therapeutically effective doses to treatany of a variety of physiological disorders. A therapeutically effectivedose refers to that amount of the compound sufficient to result in anyamelioration, impediment, prevention, or alteration of any biologicalsymptom.

b. Compounds that Bind to Bv8 and/or EG-VEGF

Systems may be designed to identify compounds capable of interactingwith (for example, binding to) or mimicking Bv8 and/or EG-VEGF, orcapable of interfering with the binding of Bv8 and/or EG-VEGF to acognate receptor, binding partner, or substrate. The compoundsidentified can be useful, for example, in modulating the activity ofwild type and/or mutant Bv8 gene products, EG-VEGF gene products, orcombination thereof; can be useful in elaborating the biologicalfunction of Bv8 and/or EG-VEGF; can be utilized in screens foridentifying compounds that disrupt normal Bv8 interactions and/orEG-VEGF interactions; or may themselves disrupt or activate suchinteractions.

The principle of the assays used to identify compounds that bind to Bv8and/or EG-VEGF, or Bv8 and/or EG-VEGF cognate receptors or substrates,involves preparing a reaction mixture of Bv8 and/or EG-VEGF and the testcompound under conditions and for a time sufficient to allow the twocomponents to interact and bind, thus forming a complex which can beremoved and/or detected in the reaction mixture. The Bv8 and/or EG-VEGFspecies used can vary depending upon the goal of the screening assay.For example, where agonists of the natural receptor are desired, thefull-length Bv8 or EG-VEGF, or a soluble truncated Bv8 or EG-VEGF, apeptide, or fusion protein containing one or more Bv8 or EG-VEGF domainsfused to a protein or polypeptide that affords advantages in the assaysystem (e.g., labeling, isolation of the resulting complex, etc.) can beutilized. Where compounds that directly interact with Bv8 and/or EG-VEGFare sought, peptides corresponding to Bv8 or EG-VEGF and fusion proteinscontaining Bv8 or EG-VEGF can be used.

The screening assays can be conducted in a variety of ways. For example,one method to conduct such an assay would involve anchoring the Bv8 orEG-VEGF polypeptide, peptide, or fusion protein thereof, or the testsubstance onto a solid phase and detecting Bv8/test compound complexesor EG-VEGF/test compound complexes anchored on the solid phase at theend of the reaction. In one embodiment of such a method, the Bv8reactant or EG-VEGF reactant may be anchored onto a solid surface, andthe test compound, which is not anchored, may be labeled, eitherdirectly or indirectly.

In practice, microtiter plates may conveniently be utilized as the solidphase. The anchored component may be immobilized by non-covalent orcovalent attachments. Non-covalent attachment may be accomplished bysimply coating the solid surface with a solution of the protein anddrying. Alternatively, an immobilized antibody, preferably a monoclonalantibody, specific for the protein to be immobilized may be used toanchor the protein to the solid surface. The surfaces may be prepared inadvance and stored.

In order to conduct the assay, the nonimmobilized component is added tothe coated surface containing the anchored component. After the reactionis complete, unreacted components are removed (e.g., by washing) underconditions such that any complexes formed will remain immobilized on thesolid surface. The detection of complexes anchored on the solid surfacecan be accomplished in a number of ways. Where the previouslynonimmobilized component is pre-labeled, the detection of labelimmobilized on the surface indicates that complexes were formed. Wherethe previously nonimmobilized component is not pre-labeled, an indirectlabel can be used to detect complexes anchored on the surface; e.g.,using a labeled antibody specific for the previously nonimmobilizedcomponent (the antibody, in turn, may be directly labeled or indirectlylabeled with a labeled anti-Ig antibody).

Alternatively, a reaction can be conducted in a liquid phase, thereaction products separated from unreacted components, and complexesdetected; e.g., using an immobilized antibody specific for a Bv8 orEG-VEGF protein, polypeptide, peptide or fusion protein or the testcompound to anchor any complexes formed in solution, and a labeledantibody specific for the other component of the possible complex todetect anchored complexes.

c. Compounds that Interfere with Bv8 and/or EG-VEGF Interactions

Macromolecules that interact with Bv8 and/or EG-VEGF are referred to,for purposes of this discussion, as “binding partners”. These bindingpartners are likely to be involved in Bv8 and/or EG-VEGF mediatedbiological pathways. Therefore, it is desirable to identify compoundsthat interfere with or disrupt the interaction of such binding partnerswhich may be useful in regulating or augmenting Bv8 activity and/orEG-VEGF activity in the body and/or controlling disorders associatedwith this activity (or a deficiency thereof).

The basic principle of the assay systems used to identify compounds thatinterfere with the interaction between Bv8 or EG-VEGF and a bindingpartner or partners involves preparing a reaction mixture containing Bv8and/or EG-VEGF, or some variant thereof, and the binding partner underconditions and for a time sufficient to allow the two to interact andbind, thus forming a complex. In order to test a compound for inhibitoryactivity, the reaction mixture is prepared in the presence and absenceof the test compound. The test compound may be initially included in thereaction mixture, or may be added at a time subsequent to the additionof Bv8 or EG-VEGF and its binding partner. Control reaction mixtures areincubated without the test compound or with a placebo. The formation ofany complexes between the Bv8 or EG-VEGF and the binding partner is thendetected. The formation of a complex in the control reaction, but not inthe reaction mixture containing the test compound, indicates that thecompound interferes with the interaction of the Bv8 or EG-VEGF and theinteractive binding partner. Additionally, complex formation withinreaction mixtures containing the test compound and normal Bv8 protein ornormal EG-VEGF protein may also be compared to complex formation withinreaction mixtures containing the test compound and a mutant Bv8 ormutant EG-VEGF, respectively. This comparison may be important in thosecases wherein it is desirable to identify compounds that specificallydisrupt interactions of mutant, or mutated, Bv8 or EG-VEGF but not thenormal proteins.

The assay for compounds that interfere with the interaction between Bv8and/or EG-VEGF and their binding partners can be conducted in aheterogeneous or homogeneous format. Heterogeneous assays involveanchoring either Bv8 or EG-VEGF, or a binding partner thereof, onto asolid phase and detecting complexes anchored on the solid phase at theend of the reaction. In homogeneous assays, the entire reaction iscarried out in a liquid phase. In either approach, the order of additionof reactants can be varied to obtain different information about thecompounds being tested. For example, test compounds that interfere withthe interaction by competition can be identified by conducting thereaction in the presence of the test substance; such as, by adding thetest substance to the reaction mixture prior to, or simultaneously with,Bv8 or EG-VEGF and the interactive binding partner. Alternatively, testcompounds that disrupt preformed complexes, e.g. compounds with higherbinding constants that displace one of the components from the complex,can be tested by adding the test compound to the reaction mixture aftercomplexes have been formed. Various formats are described briefly below.

In a heterogeneous assay system, either the polypeptide (Bv8 or EG-VEGF)or an interactive binding partner of the polypeptide, is anchored onto asolid surface, while the non-anchored species is labeled, eitherdirectly or indirectly. In practice, microtiter plates are convenientlyutilized. The anchored species may be immobilized by non-covalent orcovalent attachments. Non-covalent attachment may be accomplished simplyby coating the solid surface with a solution of the polypeptide orbinding partner and drying. Alternatively, an immobilized antibodyspecific for the species to be anchored may be used to anchor thespecies to the solid surface. The surfaces may be prepared in advanceand stored.

In order to conduct the assay, the partner of the immobilized species isexposed to the coated surface with or without the test compound. Afterthe reaction is complete, unreacted components are removed (e.g., bywashing) and any complexes formed will remain immobilized on the solidsurface. The detection of complexes anchored on the solid surface can beaccomplished in a number of ways. Where the non-immobilized species ispre-labeled, the detection of label immobilized on the surface indicatesthat complexes were formed. Where the non-immobilized species is notpre-labeled, an indirect label can be used to detect complexes anchoredon the surface; e.g., using a labeled antibody specific for theinitially non-immobilized species (the antibody, in turn, may bedirectly labeled or indirectly labeled with a labeled anti-Ig antibody).Depending upon the order of addition of reaction components, testcompounds which inhibit complex formation or which disrupt preformedcomplexes can be detected.

Alternatively, the reaction can be conducted in a liquid phase in thepresence or absence of the test compound, the reaction productsseparated from unreacted components, and complexes detected; e.g., usingan immobilized antibody specific for one of the binding components toanchor any complexes formed in solution, and a labeled antibody specificfor the other partner to detect anchored complexes. Again, dependingupon the order of addition of reactants to the liquid phase, testcompounds which inhibit complex or which disrupt preformed complexes canbe identified.

In an alternate embodiment of the invention, a homogeneous assay can beused. In this approach, a preformed complex of the polypeptide (Bv8 orEG-VEGF) and an interactive binding partner is prepared where either thepolypeptide or binding partner is labeled, but the signal generated bythe label is quenched due to formation of the complex (see, for example,U.S. Pat. No. 4,109,496 by Rubenstein that utilizes this approach forimmunoassays). The addition of a test substance that competes with anddisplaces one of the species from the preformed complex will result inthe generation of a signal above background. In this way, testsubstances that disrupt the interaction can be identified.

In a particular embodiment, a Bv8 (or EG-VEGF) fusion can be preparedfor immobilization. For example, the polypeptide (Bv8 or EG-VEGF) or apeptide fragment thereof, can be fused to a glutathione-S-transferase(GST) gene using a fusion vector, such as pGEX-5X-1, in such a mannerthat its binding activity is maintained in the resulting fusion protein.The interactive binding partner can be purified and used to raise amonoclonal antibody, using methods routinely practiced in the art anddescribed above. This antibody can be labeled with the radioactiveisotope ¹²⁵I, for example, by methods routinely practiced in the art. Ina heterogeneous assay, the fusion protein can be anchored toglutathione-agarose beads. The interactive binding partner can then beadded in the presence or absence of the test compound in a manner thatallows interaction and binding to occur. At the end of the reactionperiod, unbound material can be washed away, and the labeled monoclonalantibody can be added to the system and allowed to bind to the complexedcomponents. The interaction between the polypeptide (Bv8 or EG-VEGF) andthe interactive binding partner can be detected by measuring the amountof radioactivity that remains associated with the glutathione-agarosebeads. A successful inhibition of the interaction by the test compoundwill result in a decrease in measured radioactivity.

Alternatively, the GST fusion protein and the interactive bindingpartner can be mixed together in liquid in the absence of the solidglutathione-agarose beads. The test compound can be added either duringor after the species are allowed to interact. This mixture can then beadded to the glutathione-agarose beads and unbound material is washedaway. Again the extent of inhibition of the interaction between Bv8 andthe binding partner or EG-VEGF and the binding partner can be detectedby adding the labeled antibody and measuring the radioactivityassociated with the beads.

In another embodiment of the invention, these same techniques can beemployed using peptide fragments that correspond to the binding domainsof the polypeptide (Bv8 or EG-VEGF) and/or the interactive bindingpartner (in cases where the binding partner is a protein), in place ofone or both of the full length proteins. Any number of methods routinelypracticed in the art can be used to identify and isolate the bindingsites. These methods include, but are not limited to, mutagenesis of thegene encoding one of the proteins and screening for disruption ofbinding in a co-immunoprecipitation assay. Compensatory mutations in thegene encoding the second species in the complex can then be selected.Sequence analysis of the genes encoding the respective proteins willreveal the mutations that correspond to the region of the proteininvolved in interactive binding. Alternatively, one protein can beanchored to a solid surface using methods described above, and allowedto interact with and bind to its labeled binding partner, which has beentreated with a proteolytic enzyme, such as trypsin. After washing, arelatively short, labeled peptide comprising the binding domain mayremain associated with the solid material, which can be isolated andidentified by amino acid sequencing. Also, once the gene coding for theintracellular binding partner is obtained, short gene segments can beengineered to express peptide fragments of the protein, which can thenbe tested for binding activity and purified or synthesized.

For example, and not by way of limitation, Bv8 and/or EG-VEGF can beanchored to a solid material as described, above, by making a GST fusionprotein and allowing it to bind to glutathione agarose beads. Theinteractive binding partner can be labeled with a radioactive isotope,such as ³⁵S, and cleaved with a proteolytic enzyme such as trypsin.Cleavage products can then be added to the anchored fusion protein andallowed to bind. After washing away unbound peptides, labeled boundmaterial, representing the intracellular binding partner binding domain,can be eluted, purified, and analyzed for amino acid sequence bywell-known methods. Peptides so identified can be produced syntheticallyor fused to appropriate facilitative proteins using recombinant DNAtechnology.

H. Pharmaceutical Compositions

The Bv8 polypeptides, EG-VEGF polypeptides, and modulators thereofdescribed herein, including agonists and antagonists of Bv8 or EG-VEGF,may be employed as therapeutic agents. These polypeptides and modulatorsof the present invention can be formulated according to known methods toprepare pharmaceutically useful compositions, whereby the Bv8 and/orEG-VEGF product is combined in a mixture with a pharmaceuticallyacceptable carrier vehicle. Therapeutic formulations of Bv8, EG-VEGF, orcombinations thereof are prepared by mixing Bv8 and/or EG-VEGF havingthe desired degree of purity, preferably essentially pure, with optionalphysiologically acceptable carriers, excipients or stabilizers(Remington's Pharmaceutical Sciences, supra), in the form of lyophilizedcake or aqueous solutions. Acceptable carriers, excipients orstabilizers are nontoxic to the cell or mammal being exposed at thedosages and concentrations employed. Examples include buffers such asphosphate, citrate and other organic acids; antioxidants includingascorbic acid; low molecular weight (less than about 10 residues)polypeptides; proteins, such as serum albumin, gelatin orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone,amino acids such as glycine, glutamine, asparagine, arginine or lysine;monosaccharides, disaccharides and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugaralcohols such as mannitol or sorbitol; salt-forming counterions such assodium; and/or nonionic surfactants such as Tween, Pluronics or PEG.

Bv8 and/or EG-VEGF to be used for in vivo administration must besterile. This is readily accomplished by any method known in the art,such as filtration through sterile filtration membranes, prior to orfollowing lyophilization and reconstitution. Bv8 may be stored inlyophilized form. Therapeutic compositions of Bv8, EG-VEGF, andcombinations thereof, generally are placed into a container having asterile access port, for example, an intravenous solution bag or vialhaving a stopper pierceable by a hypodermic injection needle.

Bv8 optionally is combined with or administered in concert with othergrowth factors. For example it may be combined with EG-VEGF or VEGF.EG-VEGF optionally is combined with or administered in concert withother growth factors. For example it may be combined with Bv8 or VEGF.

Bv8, EG-VEGF, or modulators thereof, may be used with other conventionaltherapies for treating cancer, other hematological disorders,neutropenias, immunodeficiency disorders, autoimmune disorders, and thelike.

The route of administration is in accord with known methods, e.g.injection or infusion by intravenous, intraperitoneal, intracerebral,intramuscular, intraocular, intraarterial or intralesional routes,topical administration, or by sustained release systems.

Dosages and desired drug concentrations of pharmaceutical compositionsof the present invention may vary depending on the particular useenvisioned. The determination of the appropriate dosage or route ofadministration is well within the skill of an ordinary physician. Animalexperiments provide reliable guidance for the determination of effectivedoses for human therapy. Interspecies scaling of effective doses can beperformed following the principles laid down by Mordenti, J. andChappell, W. “The use of interspecies scaling in toxicokinetics” InToxicokinetics and New Drug Development, Yacobi et al., Eds., PergamonPress, New York 1989, pp. 42-96.

Where sustained-release administration of a Bv8 and/or EG-VEGFpolypeptide or modulator is desired in a formulation with releasecharacteristics suitable for the treatment of any disease or disorderrequiring administration of the Bv8 and/or EG-VEGF polypeptide ormodulator, microencapsulation of the Bv8 and/or EG-VEGF polypeptide ormodulator, or a combination thereof, is contemplated. For example, Bv8,EG-VEGF, or a combination thereof in purified form may be entrapped inmicrocapsules prepared, for example, by coacervation techniques or byinterfacial polymerization (for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively), in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, 1980, (A. Osol, Ed).

Bv8, EG-VEGF, or a combination thereof may be incorporated intosustained release preparations for therapeutic use. Suitable examples ofsustained release preparations include semipermeable polymer matrices inthe form of shaped articles, e.g. films, or microcapsules. Examples ofsustained release matrices include polyesters, hydrogels (e.g.poly(2-hydroxyethyl-methacrylate) as described by Langer et al., 1981,J. Biomed. Mater. Res., 15:167-277 and Langer, 1982, Chem. Tech.,12:98-105 or poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919,EP 58,481), copolymers of L-glutamic acid and gamma ethyl-L-glutamate(Sidman, et al., 1983, Biopolymers 22:547), non-degradable ethylenevinyl acetate (Langer, et al., supra) or degradable lactic acid-glycolicacid copolymers such as the Lupron Depot™ (injectable microspherescomposed of lactic acid-glycoloic acid copolymer and leuprolideacetate), and poly-D-(−)-3-hydroxybutyric acid (EP 133,988).

While polymers such as ethylene-vinyl acetate and lactic acid-glycolicacid enable release of molecules for over 100 days, certain hydrogelsrelease proteins for shorter time periods. When encapsulated proteinsremain in the body for a long time, they may denature or aggregate as aresult of exposure to moisture at 37° C., resulting in a loss ofbiological activity and possible changes in immunogenicity. Rationalstrategies can be devised for protein stabilization depending on themechanism involved. For example, if the aggregation mechanism isdiscovered to be intermolecular S—S bond formation throughthio-disulfide interchange, stabilization may be achieved by modifyingsulfhydryl residues, lyophilizing from acidic solutions, controllingmoisture content, using appropriate additives, and developing specificpolymer matrix compositions.

Sustained release Bv8 and/or EG-VEGF compositions also includeliposomally entrapped Bv8 and/or EG-VEGF. Liposomes containing Bv8and/or EG-VEGF are prepared by methods known in the art. (Epstein, etal., 1985, Proc. Natl. Acad. Sci. 82:3688; Hwang, et al., 1980, Proc.Natl. Acad. Sci. USA 77:4030; DE 3218121A; EP 52322A; EP 36676A; EP88046A; EP 143949A; EP 142641A; Japanese Pat. App. No. 83-118008; U.S.Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324A). Ordinarily theliposomes are of the small (about 200-800 Angstroms) unilamelar type inwhich the lipid content is greater than about 30 mol. % cholesterol, theselected proportion being adjusted for the optimal Bv8 therapy.

When applied topically, Bv8, EG-VEGF, or a combination thereof, issuitably combined with other ingredients, such as carriers and/oradjuvants. There are no limitations on the nature of such otheringredients, except that they must be physiologically acceptable andefficacious for their intended administration, and cannot degrade theactivity of the active ingredients of the composition. Examples ofsuitable vehicles include ointments, creams, gels, or suspensions, withor without purified collagen. The compositions also may be impregnatedinto transdermal patches, plasters, and bandages, preferably in liquidor semi-liquid form.

For obtaining a gel formulation, Bv8, EG-VEGF, or a combination thereofformulated in a liquid composition may be mixed with an effective amountof a water-soluble polysaccharide or synthetic polymer such as PEG toform a gel of the proper viscosity to be applied topically. Thepolysaccharide that may be used includes, for example, cellulosederivatives such as etherified cellulose derivatives, including alkylcelluloses, hydroxyalkyl celluloses, and alkylhydroxyalkyl celluloses,for example, methylcellulose, hydroxyethyl cellulose, carboxymethylcellulose, hydroxypropyl methylcellulose, and hydroxypropyl cellulose;starch and fractionated starch; agar; alginic acid and alginates; gumarabic; pullullan; agarose; carrageenan; dextrans; dextrins; fructans;inulin; mannans; xylans; arabinans; chitosans; glycogens; glucans; andsynthetic biopolymers; as well as gums such as xanthan gum; guar gum;locust bean gum; gum arabic; tragacanth gum; and karaya gum; andderivatives and mixtures thereof. The preferred gelling agent herein isone that is inert to biological systems, nontoxic, simple to prepare,and not too runny or viscous, and will not destabilize the Bv8 and/orEG-VEGF held within the gel.

Preferably the polysaccharide is an etherified cellulose derivative,more preferably one that is well defined, purified, and listed in USP,e.g., methylcellulose and the hydroxyalkyl cellulose derivatives, suchas hydroxypropyl cellulose, hydroxyethyl cellulose, and hydroxypropylmethylcellulose. Most preferred herein is methylcellulose.

The polyethylene glycol useful for gelling is typically a mixture of lowand high molecular weight PEGs to obtain the proper viscosity. Forexample, a mixture of a PEG of molecular weight 400-600 with one ofmolecular weight 1500 would be effective for this purpose when mixed inthe proper ratio to obtain a paste.

The term “water soluble” as applied to the polysaccharides and PEGs ismeant to include colloidal solutions and dispersions. In general, thesolubility of the cellulose derivatives is determined by the degree ofsubstitution of ether groups, and the stabilizing derivatives usefulherein should have a sufficient quantity of such ether groups peranhydroglucose unit in the cellulose chain to render the derivativeswater-soluble. A degree of ether substitution of at least 0.35 ethergroups per anhydroglucose unit is generally sufficient. Additionally,the cellulose derivatives may be in the form of alkali metal salts, forexample, the Li, Na, K, or Cs salts.

If methylcellulose is employed in the gel, preferably it comprises about2-5%, more preferably about 3%, of the gel and Bv8 and/or EG-VEGF ispresent in an amount of about 300-1000 mg per ml of gel.

Semipermeable, implantable membrane devices are useful as means fordelivering drugs in certain circumstances. For example, cells thatsecrete Bv8 and/or EG-VEGF, Bv8 and/or EG-VEGF variants, Bv8 and/orEG-VEGF chimeras, or agonists or antagonists of Bv8 and/or EG-VEGF canbe encapsulated, and such devices can be implanted into a patient.Accordingly, also included is a method for preventing or treatingcancer, other hematological disorders, neutropenias, immunologicaldisorders, autoimmune disorders, and the like, that comprises implantingcells that secrete Bv8 and/or EG-VEGF, or agonists or antagoniststhereof as may be required for the particular condition, into the bodyof patients in need thereof Finally, the present invention includes adevice for preventing or treating cancer, other hematological disorders,neutropenias, immunodeficiency disorders, autoimmune disorders, and thelike, by implantation into a patient of an implant comprising asemipermeable membrane, and cells that secrete Bv8 and/or EG-VEGF (oragonists or antagonists thereof as may be required for the particularcondition) encapsulated within said membrane and said membrane beingpermeable to Bv8 and/or EG-VEGF (or agonists or antagonists thereof) andimpermeable to factors from the patient detrimental to the cells. Thepatient's own cells, transformed to produce Bv8 and/or EG-VEGF ex vivo,could be implanted directly into the patient, optionally without suchencapsulation. The methodology for the membrane encapsulation of livingcells is familiar to those of ordinary skill in the art, and thepreparation of the encapsulated cells and their implantation intopatients may be accomplished without undue experimentation.

The pharmaceutical composition comprising Bv8, EG-VEGF, or a combinationthereof, or agonists or antagonists thereof, is preferably located in asuitable container. The container is preferably accompanied byinstructions detailing the appropriate use and dosage of thepharmaceutical composition. One skilled in the art will recognize thatthese instructions will vary depending upon the method of treatment.

I. Methods of Treatment

Bv8, EG-VEGF, and their agonists and antagonists provided herein may beused in a number of diagnostic assays and treatments. Bv8 and/or EG-VEGFinduce proliferation of bone marrow cells and their progeny, includingbut not limited to, hematopoietic stem cells, CD34+ myeloid progenitorcells, CD34+ lymphoid progenitor cells, myeloid precursor cells,lymphoid precursor cells, monocytes, and lymphocyes. This proliferationleads to an increase in the number of white blood cells, including Bcells, T cells, macrophages, and in particular, neutrophils.

Bv8, EG-VEGF, and their agonists are therapeutically useful, forexample, in the treatment of disorders or conditions where it isdesirable to increase proliferation of bone marrow cells and theirprogeny, including but not limited to myeloid progenitor cells, myeloidprecursor cells, neutrophils, lymphoid progenitor cells, lymphoidprecursor cells, and lymphocytes, or enhance the survival of particulartypes of blood cells, such as neutrophils, B cells, or T cells. In anembodiment, Bv8, EG-VEGF, or an agonist thereof is administered to amammal in an amount effective to treat the disease or disorder.Preferably, the mammal is a human. Bv8, EG-VEGF, or a combinationthereof may be administered in a polypeptide or nucleic acid form.

For example, Bv8, EG-VEGF, and their agonists are therapeutically usefulfor treating conditions and disorders associated with neutropenia,lymphopenia, or immunodeficiency disorders, which may be primary orsecondary immunodeficiency disorders. These conditions and disorders maybe associated, for example, with genetic disorders, B cell deficiencies,T cells deficiencies, infectious diseases including bacterial and viralinfection, infiltrative and hematological disorders, surgery and trauma,and administration of a therapeutic agent that has a secondaryimmunosuppressive effect.

In some instances the secondary immunosuppressive effect results in animmunodeficiency disorder. Bv8, EG-VEGF, or a combination thereof may beused to promote hematopoietic recovery from myelosuppression, forexample, in cancer patients undergoing therapeutic treatments whereinthe therapeutic agents severely lower the level of circulatingleukocytes and compromise the patient's immune system. Bv8, EG-VEGF, ora combination thereof may be administered prior to, in combination with,or subsequent to radiation, high dose chemotherapy, or other anti-cancerdrugs to promote hematopoietic recovery and/or increase the number ofcirculating neutrophils, B cells, and T cells.

Bv8, EG-VEGF, or a combination thereof may be administered with anothercompound or composition. Bv8 and/or EG-VEGF may be administered priorto, during, or after treatment with the compound or composition suchthat the therapeutic efficacy of Bv8, EG-VEGF, and/or the compound orcomposition is increased. As described above, the compound orcomposition may be a chemotherapeutic agent. Preferred chemotherapeuticagents include but are not limited to vincristine, cisplatin, oxoplatin,methotrexate, 3′-azido-3′-deoxythymidine, taxanes (e.g. paclitaxel(TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.J.), and doxetaxel(TAXOTERE®, Rhône-Poulenc Rorer, Antony, France)) and/or anthracyclineantibiotics. The manufacturers' instructions may be followed indetermining the preparation and dosing schedules for suchchemotherapeutic agents or they may be determined empirically by theskilled practitioner. Preparation and dosing schedules for suchchemotherapy are also described in Chemotherapy Service Ed., M.C. Perry,Williams & Wilkins, Baltimore, Md. (1992).

In another embodiment, Bv8, EG-VEGF, or a combination thereof isadministered with VEGF or an agonist thereof The VEGF may be a receptorselective mutant of VEGF. In one embodiment the compound is a FLT1receptor selective mutant of VEGF or an agonist thereof. In anotherembodiment, the compound is a KDR receptor selective mutant of VEGF oran agonist thereof.

Antagonists of Bv8 and EG-VEGF and combinations thereof may betherapeutically useful for treating hematological disorders associatedwith abnormal proliferation or differentiation of bloods. The abnormalproliferation or differentiation may result in dysplastic changes inbloods cells and hematological malignancies. Specific embodimentsinclude treating leukemia, myeloproliferative disorders, myelodysplasticsyndromes, lymphoproliferative disorders, and lymphodysplastic disordersin a patient. Cells in various leukemic disorders, such as ALL, AML,MPD, CML, and MDS cells, have been found to express receptors for Bv8and EG-VEGF. Antagonists of Bv8 and EG-VEGF are useful to inhibitproliferation of these leukemic cells.

Bv8 and EG-VEGF induce B and T cell activation. Agonists and antagonistsof these molecules are therapeutically useful to modulate an immuneresponse. Bv8, EG-VEGF or a combination thereof or agonists thereof maybe administered to induce proliferation and/or activation of Blymphocytes, CD4+ T lymphocytes, and/or CD8+ T lymphocytes. Antagonistsof Bv8 and EG-VEGF may be administered to inhibit proliferation and/oractivation of B lymphocytes, CD4+ T lymphocytes, and/or CD8+ Tlymphocytes.

Bv8 and/or EG-VEGF may promote or inhibit proliferation and activationof CD4+ T lymphocytes. Bv8 and EG-VEGF induce cytokine production inCD4+ T lymphocytes. In an embodiment, the cytokines are IL-2 and/orIFN-γ. Bv8 or EG-VEGF agonists that selectively induce IL-2 synthesis inCD4+ T lymphocytes may be useful to induce proliferation of CD4+ Tcells. Bv8 or EG-VEGF agonists that selectively induce IFN-γ synthesisin CD4+ T lymphocytes by may be useful to inhibit proliferation of CD4+T cells.

Bv8 antagonists, EG-VEGF antagonists, and combinations thereof may beuseful to treat autoimmune disorders where a decrease in the number ofactivated B cells, CD4+ T cells, and/or CD8+ T cells is desirable.Specific embodiments include using the agents and compositions providedherein to treat type II, III, and IV hypersensitivity responsesassociated with autoimmune disorders. In a preferred embodiment, theautoimmune disorder is inflammatory bowel disease, Crohn's disease,colitis, or graft versus host disease.

Compounds such as those identified by the screening assays in section 4and 5, above, may be used to modulate the level of Bv8 and/or EG-VEGFactivity or expression. Specifically, compounds identified that are Bv8agonists and /or EG-VEGF or that are able to stimulate the binding ofBv8 and/or EG-VEGF to its receptors may be useful for treatments whereinan increased level of Bv8 and/or EG-VEGF activity is desired. Similarly,compounds identified that are able to increase Bv8 gene expression,EG-VEGF gene expression, or a combination thereof, may be useful forthis type of treatment. Bv8 agonists and/or EG-VEGF agonists andcompounds that increase Bv8 and/or EG-VEGF gene expression may be usefulto treat neutropenia, lymphopenia, and immunodeficiency disorders, wherean increase in the number of neutrophils, B lymphocytes, and/or Tlymphocytes is desirable.

Compounds, such as Bv8 antagonists and/or EG-VEGF antagonists identifiedby the screening assays in section 4 and 5 above, may be used tomodulate the level of Bv8 and/or EG-VEGF activity or expression,respectively, as described herein. Compounds, such as Bv8 agonistsand/or EG-VEGF agonists identified by the screening assays in section 4and 5 above, may be useful to modulate an immune response as describedherein.

It is understood that the methods of increasing bone marrow cellproliferation and inhibiting bone marrow cell proliferation can beperformed in vivo or in vitro. In some cases, it may be desirable to addBv8, EG-VEGF, or a combination thereof to a cell sample in vitro so asto stimulate proliferation of a specific cell type. The sample treatedwith Bv8, EG-VEGF, or a combination thereof can then be used inscreening assays or be transplanted into an individual in need oftreatment or into an animal model.

An effective amount of Bv8 or a Bv8 agonist or antagonist, EG-VEGF or anagonist or antagonist thereof, to be employed therapeutically willdepend, for example, upon the therapeutic objectives, the route ofadministration, and the condition of the patient. Accordingly, it willbe necessary for the therapist to titer the dosage and modify the routeof administration as required to obtain the optimal therapeutic effect.Typically, the clinician will administer the Bv8 and/or EG-VEGF until adosage is reached that achieves the desired effect. A typical dailydosage for systemic treatment may vary from about 10 ng/kg to up to 100mg/kg of mammal body weight or more per day, preferably about 1μg/kg/day to 10 mg/kg/day, depending upon the route of administration.It is anticipated that different formulations will be effective fordifferent treatment compounds and different disorders, and thatadministration targeting one organ or tissue, for example, maynecessitate delivery in a manner different from that to another organ ortissue.

Alternatively, Bv8 and/or EG-VEGF is formulated and delivered to thetarget site or tissue at a dosage capable of establishing in the tissuea level of Bv8 and/or EG-VEGF that is efficacious but not unduly toxic.This intra-tissue concentration should be maintained if possible bycontinuous infusion, sustained release, topical application, cellimplant expressing Bv8 and/or EG-VEGF, or injection at empiricallydetermined frequencies. The progress of this therapy is easily monitoredby conventional assays.

The dosing regimen must be determined based on the individualcircumstances. However, in a preferred embodiment, Bv8 and/or EG-VEGF,or an agonist or antagonist thereof, is administered every day, morepreferably every other day and even more preferably at least two times aweek. The treatment is preferably continued for six months, morepreferably for one month and even more preferably for at least twoweeks. One skilled in the art will appreciate that the exact dosingregimen must be determined by the therapist based on the individualcircumstances.

Polynucleotides encoding Bv8 and/or EG-VEGF polypeptide may also be usedin gene therapy. In gene therapy applications, genes are introduced intocells in order to achieve in vivo synthesis of a therapeuticallyeffective genetic product, for example for replacement of a defectivegene. “Gene therapy” includes both conventional gene therapy where alasting effect is achieved by a single treatment, and the administrationof gene therapeutic agents, which involves the one time or repeatedadministration of a therapeutically effective DNA or mRNA. AntisenseRNAs and DNAs can be used as therapeutic agents for blocking theexpression of certain genes in vivo. It has already been shown thatshort antisense oligonucleotides can be imported into cells where theyact as inhibitors, despite their low intracellular concentrations causedby their restricted uptake by the cell membrane. (Zamecnik et al., 1986,Proc. Natl. Acad. Sci. USA 83:4143-4146). The oligonucleotides can bemodified to enhance their uptake, e.g. by substituting their negativelycharged phosphodiester groups by uncharged groups.

There are a variety of techniques available for introducing nucleicacids into viable cells. The techniques vary depending upon whether thenucleic acid is transferred into cultured cells in vitro, or in vivo inthe cells of the intended host. Techniques suitable for the transfer ofnucleic acid into mammalian cells in vitro include the use of liposomes,electroporation, microinjection, cell fusion, DEAE-dextran, the calciumphosphate precipitation method, etc. The currently preferred in vivogene transfer techniques include transfection with viral (typicallyretroviral) vectors and viral coat protein-liposome mediatedtransfection (Dzau et al., 1993, Trends in Biotechnology 11, 205-210).In some situations it is desirable to provide the nucleic acid sourcewith an agent that targets the target cells, such as an antibodyspecific for a cell surface membrane protein or the target cell, aligand for a receptor on the target cell, etc. Where liposomes areemployed, proteins which bind to a cell surface membrane proteinassociated with endocytosis may be used for targeting and/or tofacilitate uptake, e.g. capsid proteins or fragments thereof tropic fora particular cell type, antibodies for proteins which undergointernalization in cycling, proteins that target intracellularlocalization and enhance intracellular half-life. The technique ofreceptor-mediated endocytosis is described, for example, by Wu et al.,1987, J. Biol. Chem. 262, 4429-4432; and Wagner et al., 1990, Proc.Natl. Acad. Sci. USA 87, 3410-3414. For review of gene marking and genetherapy protocols see Anderson et al., 1992, Science 256, 808-813.

Peptide or nucleic acid sequences encoding Bv8 and/or EG-VEGF sequencecan also be used in methods of diagnosis. Abnormal expression of Bv8and/or EG-VEGF may indicate abnormalities in hematopoiesis, onset ofhematological disorders, onset of neutropenia, onset of immunodeficiencydisorders, or onset of autoimmune disorders. Moreover, a sample from apatient may be analyzed for mutated or dysfunctional Bv8 and/or EG-VEGF.Generally, such methods include comparing Bv8 expression and/or EG-VEGFexpression in a sample from a patient to that of a control.

J. Articles of Manufacture

In another aspect the invention contemplates an article of manufacturecomprising materials useful for the treatment or prevention of a diseaseor disorder. The article of manufacture preferably comprises a containerand a label or package insert on or associated with the container.Suitable containers include, for example, bottles, vials, syringes etc.The containers may be formed from a variety of materials such as glassand plastic. The container holds a composition comprising Bv8 and/orEG-VEGF, or an agonist or antagonist thereof, and the label or packageinsert preferably provides instructions for using the Bv8 and/orEG-VEGF, or an agonist or antagonist thereof. In one embodiment, thearticle of manufacture comprises a Bv8 antagonist and/or EG-VEGFantagonist, and instructions for using the antagonist to treat orprevent hematological disorders such as leukemia myeloproliferativedisorders, myelodysplastic disorders, and the like. In anotherembodiment, the article of manufacture comprises Bv8 and/or EG-VEGF andinstructions for using the polypeptide to treat or prevent a conditionthat is associated with abnormal hematopoiesis. In yet anotherembodiment, the article of manufacture comprises Bv8 and/or EG-VEGF andinstructions for using the polypeptide to treat an immunodeficiencydisorder. The package insert may also indicate the appropriate dosingregimen. In one embodiment the insert indicates that the composition isto be administered in a dose of between about 0.01 μg/kg and 50 mg/kg.

EXAMPLES

Commercially available reagents referred to in the examples were usedaccording to manufacturer's instructions unless otherwise indicated. Thesource of those cells identified in the following examples, andthroughout the specification, by ATCC accession numbers is the AmericanType Culture Collection, Manassas, Va.

All references cited herein are hereby incorporated by reference.

Example 1 Expression Analyses of Bv8 and Its Receptors

To elucidate the expression pattern of Bv8, Dot blot analysis wasperformed on RNA arrays representing a panel of tissues and cell lines.from a wide variety of human, mouse and rat tissues. Tissue/cell RNAarrays and blots were purchased from CLONTECH. cDNA probes were preparedusing 50 ng of human or mouse Bv8 coding sequence with methods describedpreviously (LeCouter et al., 2001, Nature). As shown in FIG. 13, Bv8expression appeared restricted to peripheral blood leukocytes, bonemarrow tissues and the previously characterized testis (LeCouter et al.,2003, PNAS).

To identify the cell types that express hBv8, in situ hybridization(ISH) experiments were performed using a series of inflammatory tissuespecimens, in which increased level of leukocytes were present. Tissueswere processed for ISH and [³³P]UTP-labeled RNA probes were generatedusing standard methods and materials. Sense and antisense probes forhBv8 were synthesized from a cDNA fragment corresponding to nucleotides533-1132; mBv8, nucleotides 886-1611; and mR-1, nucleotides 220-946.This probe is 81.2% identical to mR-2. Within both the inflammatorytonsils and the inflammatory appendix, strong hybridization signals ofBv8 were detected mainly in neutrophils and associated infiltratingcells (FIG. 14).

To confirm the cellular expression pattern of Bv8 and its receptors,real time quantitative PCR analyses (Heid et al., 1996, Genome Res.,6:986-994) were performed in hematopoietic cells and leukemia celllines. Total RNA was prepared from human and mouse hematopoietic stemcells, lineage-committed progenitor cells or leukemia cell lines usingthe RNeasy kit and following the manufacturer's instructions. Human cellpreparations were purchased from AllCells Inc. (Berkeley, Calif.),lineage committed mouse cells and stem cells (Sca+c-Kit+) were preparedfrom a pool of 28 femurs and sorted as previously described (Gerber,2002, Nature). Human cell lines (HL-60, K562, Hel-92, TF-1 and KG-1)were obtained from the ATCC. For real-time RT-PCR analyses, 100 ng oftotal RNA was used. For both mouse and human samples, standard curveswere generated using testis RNA. Primers and probes used in the analysiswere as follows:

Human (h) Forward 5′ TGGGCTACACTGAGCACCAG 3′ SEQ ID NO:11 GADPH Reverse5′ CAGCGTCAAAGGTGGAGGAG 3′ SEQ ID NO:12 Probe 5′ FAM- SEQ ID NO:13TGGTCTCCTCTGACTTCAACAGCGCAC- TAMRA 3′ Human (h) Forward 5′CCATTTTTTGGGCGGAGG 3′ SEQ ID NO:14 Bv8 Reverse 5′ CCGTAAACAGGCCAAGCCT 3′SEQ ID NO:15 Probe 5′ FAM- SEQ ID NO:16 TGCATCACACTTGCCCATGTCTG C- TAMRA3′ hEG- Forward 5′ CCGGCAGCCACAAGGTC 3′ SEQ ID NO:17 VEGF Reverse 5′TGGGCAAGCAAGGACAGG 3′ SEQ ID NO:18 Probe 5′ FAM- SEQ ID NO:19CCTTCTTCAGGAAACGCAAGCACCAC- TAMRA 3′ hBv8/EG- Forward 5′GGCGCCCTTCTACGGCT 3′ SEQ ID NO:20 VEGF Reverse 5′ TCTCCTTCACGAACACGGTG3′ SEQ ID NO:21 Receptor-1 Probe 5′ FAM-CACCATCGTGCGCGACTTCTTCC- SEQ IDNO:22 TAMRA 3′ hBv8/EG- Forward 5′ GGAAATGACATCTGTGTTCATGC 3′ SEQ IDNO:23 VEGF Reverse 5′ TCATTGTATGTTACGACTTTGCAGC 3′ SEQ ID NO:24Receptor-2 Probe 5′ FAM-CCCGTGCCCTCAAGAAGCCGA- SEQ ID NO:25 TAMRA 3′Mouse (m) Forward 5′ ATGTTCCAGTATGACTCCACTCACG 3′ SEQ ID NO:26 GADPHReverse 5′ GAAGACACCAGTAGACTCCACGACA 3′ SEQ ID NO:27 Probe 5′ FAM- SEQID NO:28 AAGCCCATCACCATCTTCCAGGAGCGAGA- TAMRA 3′ Mouse (m) Forward 5′CGGAGGATGCACCACACC 3′ SEQ ID NO:29 Bv8 Reverse 5′CCGGTTGAAAGAAGTCCTTAAACA 3′ SEQ ID NO:30 Probe 5′FAM-CCCCTGCCTGCCAGGCTTGG- SEQ ID NO:31 TAMRA 3′ mEG- Forward 5′TGAGGAAACGCCAACACCAT 3′ SEQ ID NO:32 VEGF Reverse 5′ CCGGGAACCTGGAGCAC3′ SEQ ID NO:33 Probe 5′ FAM-CCTGTCCCTGCTCACCCAGCCTG- SEQ ID NO:34 TAMRA3′ mBv8/EG- Forward 5′ CAGCGCACATGAAGACTTG 3′ SEQ ID NO:35 VEGF Reverse5′ GTCATCTTCGGTTTCCTGAGT 3′ SEQ ID NO:36 Receptor-1 Probe 5′FAM-TCCAGGCAGCACCCCTGATG- SEQ ID NO:37 TAMRA 3′ mBv8/EG- Forward 5′GAACTCCACGTGAGCGCA 3′ SEQ ID NO:38 VEGF Reverse 5′ GGGTGCCATGTTGATGATGCT3′ SEQ ID NQ:39 Receptor-2 Probe 5′ FAM- SEQ ID NO:40GTCCCTGATACACACCAGCCCACCTG- TAMRA 3′

As shown in FIGS. 15A-D, hBv8 mRNA is most prominently expressed in bonemarrow and testis, whereas placenta expresses about 10% of the level intestis (FIG. 15A). Among different hematopoietic cell types, hBv8 mRNAis prominently expressed in neutrophils, with moderate expressions inmonocytes and bone marrow-mononuclear cells as well (FIG. 15B). Incomparison, both the Bv8/EG-VEGF receptor 1 and receptor 2 are expressedat high levels in CD34+ cells, AC133 and BM-MNCs, but not in neutrophilsor monocytes (FIGS. 15C,D).

Expression of hBv8 and hBv8/EG-VEGF receptors was also studied invarious leukemia cell lines using real time quantitative PCR analysis.As shown in FIG. 16, certain leukemia cell lines (such as HL60 CML, K562CML and KG-1 AML) express significant levels of both Bv8 and itsreceptors.

Example 2 Colony Formation Assays

The biological functions of Bv8 in mouse and human hematopoietic cellswere studied using a methylcellulose colony formation assay. Mouse bonemarrow mononuclear cells were collected by flushing the femurs of micewith 3 ml of cold (4 C) Iscove's MDM containing 20% FCS. Red blood cellswere lysed with 10 mM NH₄Cl in 10 mM Tris pH7.2 on ice for 10 minutes.The remaining mononuclear cells were washed in media and pelleted bycentrifugation. For methylcellulose cultures, 70000 cells were plated oneach 35 mm grided-plate in media preparations (MethoCult M3434, acomplete media containing SCF, IL-3, IL-6 and Epo and M3334, a basalmedia containing Epo alone) all purchased from StemCell TechnologiesInc. according to the manufacturer's instructions. Mouse IL-3, IL-6 andSCF were purchased from StemCell Technologies Inc., and VEGF, EG-VEGFand Bv8 were produced at Genentech as previously described (LeCouter etal., 2001, Nature, 412:877-884; LeCouter et al., 2003, Proc. Natl. Acad.Sci. USA, 100:2685-2690). Final concentrations of exogenous factors wereas follows: 10 ng/ml mouse IL-3, 10 ng/ml mouse IL-6, 50 ng/ml mouseSCF, 10 ng/ml VEGF, 5 or 50 nM EG-VEGF or 50 nM mouse Bv8. After 12 daysin culture at 37 C and 5% CO2, hematopoietic colonies were enumeratedfor triplicate samples using a light microscope.

As shown in FIG. 17A, addition of Bv8 at either 5 nM or 50 nMsignificantly increased the number of colonies formed in mouse BM-MNCs.

For human cultures, 70000 bone marrow derived mononuclear cells(AllCells Inc) were plated in MethoCult 4434 complete, 4330 basal media,or basal media supplemented with 10 ng/ml IL-3, 10 ng/ml IL-6, 5 ng/mlG-CSF, 5 ng/ml GM-CSF, 50 nM EG-VEGF, and 50 nM Bv8, as indicated (allfrom StemCell Technologies Inc.). Cell colonies were identified andcounted by microscopic observation following 14-16 days in culture.

As shown in FIG. 17B, Bv8 or EG-VEGF increased colony formations of atleast certain types of lineage-committed myeloid progenitor cells. Forexample, when Bv8 or EG-VEGF were added to the basal media supplementedwith IL-3 and IL-6, colony numbers for CFU-GM cells was increased byabout 1.7-fold and about 2.2-fold, respectively; CFU-G cells by about1.7-fold and about 2.5-fold, respectively; and CFU-GEMM cells by about4-fold and about 7-fold, respectively. It is worth of noticing that, interms of colony numbers, these increases resembled the groups treatedwith G-CSF, a well known granulocyte colony-stimulating factor.

Example 3 Cell Mobilization Assays

Immunodeficient (nude) mice were injected via the tail vein withAdenovirus encoding LacZ (5×10⁸ pfu), VEGF (10⁷ pfu), EG-VEGF (5×10⁸pfu) or Bv8 (5×10⁸ pfu). This route of administration was employed toachieve systemic production of the secreted factors. To assess HSCmobilization, blood samples were collected from the retro-orbital sinusat days 3, 6 and 12, and differential blood cell counts were determinedusing a Coulter counter. At necropsy sessions on days 6 and 12, bodyweight and organ weights were determined. Bv8 enhanced the survival ofBBC epithelial cells. In particular, fewer apoptotic cells were visiblein culture in the presence of either concentration of Bv8 than in thepresence of 2% FCS or 25 nM EG-VEGF. Bv8 and VEGF showed a synergisticeffect, with a combination of the two compounds increasing cell survivalto a greater extent than either growth factor on its own or 10% FCS.

As shown in FIG. 18, adenoviral-Bv8 injection resulted in increase ofwhite blood cell counts by about 2 to about 2.5 fold. Meanwhile, thecounts of red blood cells or platelets did not have any significantchanges. Similar effects can be seen when EG-VEGF expression wasintroduced by adenoviral injection.

Example 4 Bv8/EG-VEGF Receptor Expression in Lymphocytes

Expression of Bv8/EG-VEGF and their respective receptors by human andmurine lymphocytes was studied using Real time quantitative PCRanalysis. Acutely isolated human and mouse purified cells and humancells from commercially available preparations (AllCells Inc., Berkley,Calif.) were analyzed for receptor expression. B cells, CD4⁺ T cells,CD8⁺ T cells, and natural killer cells were positively selected using anappropriate antibody (anti-CD19, anti-CD4, anti-CD8, and anti-CD56antibodies respectively) conjugated to paramagnetic beads (MiltenyiBiotec, Auburn, Calif.). Briefly, 10⁷ total cells in 90 μl of MACSbuffer (PBS with 0.5% bovine serum albumin and 2 mM EDTA) and 10 μlantibody-conjugated paramagnetic beads were incubated at 4° C. for 15minutes. The beads were then washed in an excess of MACS buffer,centrifuged at 300×g for 10 minutes, and the pellet resuspended in 2 mlof MACS buffer. The cell suspension was then applied to a LS+/VS+selection column (Miltenyi Biotec, Auburn, Calif.) that had been placedin the magnetic field of a MACS separator (Miltenyi Biotec, Auburn,Calif.). The column was rinsed two times with 3 ml of MACS buffer,removed from the separator, and the positive cell fraction flushed fromthe column with MACS buffer using the plunger supplied with the column.

Total RNA was prepared from positively selected cells using a RNeasy kit(Qiagen, Valencia, Calif.) following the manufacturer's instructions. Atleast two independently purified human and mouse RNA isolates wereanalyzed in these studies with similar results. For real timequantitative PCR analysis, 50 ng of total RNA served as template forreactions that assessed expression of EG-VEGF, Bv8, and their cognatereceptors Bv8/EG-VEGF receptor-1 and Bv8/EG-VEGF receptor-2 (LeCouter etal., 2003, Proc. Natl. Acad. Sci USA, 100:2685-2690). For both mouse andhuman samples, standard curves were generated using testis RNA. Primersand probes used in the analysis were as follows:

Human (h) Forward 5′ CCATTTTTTGGGCGGAGG 3′ SEQ ID NO:14 Bv8 Reverse 5′CCGTAAACAGGCCAAGCCT 3′ SEQ ID NO:15 Probe 5′ FAM-TGCATCACACTTGCCCATGTCTGC- SEQ ID NO:16 TAMRA 3′ hEG- Forward 5′ CCGGCAGCCACAAGGTC SEQ ID NO:17VEGF Reverse 5′ TGGGCAAGCAAGGACAGG SEQ ID NO:l8 Probe 5′FAM-CCTTCTTCAGGAAACGCAAGCACCAC- SEQ ID NO:19 TAMRA 3′ hBv8/EG- Forward5′ GGCGCCCTTCTACGGCT 3′ SEQ ID NO:20 VEGF Reverse 5′TCTCCTTCACGAACACGGTG 3′ SEQ ID NO:21 Receptor-1 Probe 5′FAM-CACCATCGTGCGCGACTTCTTCC- SEQ ID NO:22 TAMRA 3′ hBv8/EG- Forward 5′GGAAATGACATCTGTGTTCATGC 3′ SEQ ID NO:23 VEGF Reverse 5′TCATTGTATGTTACGACTTTGCAGC 3′ SEQ ID NO:24 Receptor-2 Probe 5′FAM-CCCGTGCCCTCAAGAAGCCGA- SEQ ID NO:25 TAMRA 3′ Mouse (m) Forward 5′CGGAGGATGCACCACACC 3′ SEQ ID NO:29 Bv8 Reverse 5′CCGGTTGAAAGAAGTCCTTAAACA 3′ SEQ ID NO:30 Probe 5′FAM-CCCCTGCCTGCCAGGCTTGG- SEQ ID NO:31 TAMRA 3′ mEG- Forward 5′TGAGGAAACGCCAACACCAT 3′ SEQ ID NO:32 VEGF Reverse 5′ CCGGGAACCTGGAGCAC3′ SEQ ID NO:33 Probe 5′ FAM-CCTGTCCCTGCTCACCCAGCCTG- SEQ ID NO:34 TAMRA3′ mBv8/EG- Forward 5′ CAGCGCACATGAAGACTTG 3′ SEQ ID NO:35 VEGF Reverse5′ GTCATCTTCGGTTTCCTGAGT 3′ SEQ ID NO:36 Receptor-1 Probe 5′FAM-TCCAGGCAGCACCCCTGATG- SEQ ID NO:37 TAMRA 3′ mBv8/EG- Forward 5′GAACTCCACGTGAGCGCA 3′ SEQ ID NO:38 VEGF Reverse 5′ GGGTCCCATGTTGATGATGCT3′ SEQ ID NO:39 Receptor-2 Probe 5′ FAM-CTCCCTGATACACACCA SEQ ID NO:40GCCCACCTG-TAMRA 3′

As shown in FIGS. 19A-D, human and mouse B cells, CD4⁺ T cells, CD8⁺ Tcells, and natural killer cells express Bv8/EG-VEGF receptor-1 andreceptor-2. These results indicate that these lymphocytes may beresponsive in vivo to Bv8 and/or EG-VEGF ligand, which is primarilyexpressed by neutrophils.

Example 5 B Cell Proliferation Promoted by Bv8/EG-VEGF

The ability of Bv8 and/or EG-VEGF to promote the proliferation of Bcells was studied. B cells were isolated from the spleens of Balb/C orC57B1/6J mice. Spleens were harvested from the mice and mechanicallydissociated between two glass slides. The cell preparation was suspendedin MACS buffer (PBS with 0.5% bovine serum albumin and 2 mM EDTA) andthen passed through a 40 μm nylon cell strainer. The resultant singlecell suspension was applied to a Ficoll gradient (Lymphocyte M,Cedarlane Laboratories Ltd., Ontario, Canada) and centrifuged at 2500rpm for 15 minutes. The cells at the interface were collected and washedthree times in MACS buffer. B cells were then positively selected fromthe purified cell population using CD19 Microbeads (Miltenyi Biotec,Auburn, Calif.) as described above for Example 1.

For proliferation assays, 5×10⁵ cells per well were plated in aflat-bottom 96-well plate in RPMI assay media (RMPI 1640 supplementedwith 10% fetal calf serum and penicillin/streptomycin solution (LifeTechnologies, Inc., Rockville, Md.). Recombinant Bv8 or EG-VEGF wasadded to each well at a concentration ranging from 2 to 200 nM. In somewells, 20 or 30 μg/ml of anti-mouse IgM Fab fragment (JacksonImmunoResearch, West Grove, Pa.) was added to induce basal activation ofthe B cells. In the positive controls, 1 μg/ml LPS (Sigma-Aldrich, St.Louis, Mo.) or 10 μg/ml Bly (Genentech, Inc., South San Francisco,Calif.) was added to each well in lieu of recombinant Bv8 and EG-VEGF.The cells were incubated at 37° C. for 72 hours and then pulsed with 1μcurie of 3H-thymidine per ml (Amersham Biosciences, Piscataway, N.J.).Samples were collected using a Filtermate 196 Harvester (Packard,Boston, Mass.) and analyzed using a Microplate Scintillation Counter(Packard, Boston, Mass.).

Proliferation of mouse B cells was analyzed by assessing 3H-thymidineincorporation. As shown in FIG. 20, each of Bv8 and EG-VEGF reproduciblyinduced a 4 to 6 fold increase in 3H-thymidine incorporation in B cells.This data indicates that Bv8 and EG-VEGF function as mitogens andsurvival factors for B lymphocytes.

Example 6 T Cell Proliferation Promoted by Bv8/EG-VEGF

The ability of Bv8 and/or EG-VEGF to promote the proliferation of CD4⁺ Tcells was studied. CD4⁺ T cells were isolated from spleens of Balb/C orC57B1/6J mice. Spleens were harvested and mechanically dissociated asdescribed above for Example 5. The cell preparation was then suspendedin MACS buffer and purified as described above for Example 5. T cellswere negatively selected from the purified cell population using a CD4⁺T Cell Isolation Kit (Miltenyi Biotec, Auburn, Calif.).

Briefly, 10⁷ total cells in 90 μl of MACS buffer (PBS with 0.5% bovineserum albumin and 2 mM EDTA) and 10 μl MACS Anti-Hapten Microbeads wereincubated at 4° C. for 10 minutes. The beads were then washed in anexcess of MACS buffer, centrifuged at 300×g for 10 minutes, and thepellet resuspended in 2 ml of MACS buffer. The cell suspension was thenapplied to a LS+/VS+ selection column (Miltenyi Biotec, Auburn, Calif.)that had been placed in the magnetic filed of a MACS separator (MiltenyiBiotec, Auburn, Calif.). The effluent from the column, representing theenriched CD4⁺ T cell fraction, was collected. The column was rinsed fourtimes with 3 ml of MACS buffer and the effluent representing theenriched CD4⁺ T cell fraction was collected. The enriched CD4+ T cellfraction was washed in MACS buffer and then resuspended in RPMI assaymedia.

Proliferation assays employing the negatively selected CD4+ T cells wereperformed as described above for Example 5. Recombinant Bv8 or EG-VEGFwas added to each well at a concentration ranging from 2 to 200 nM. Somewells were precoated for 2 hours at 37° C. with 0.5 μg/ml anti-mouse CD3antibody (Pharmingen, LaJolla, Calif.) and/or 1 μg/ml anti-mouse CD28antibody (Pharmingen, LaJolla, Calif.) in carbonate buffer, pH 9.0. Theantibodies crosslinked the respective receptors on the surface of the Tcells, inducing basal activation. Addition of anti-CD3 antibodies incombination with anti-CD28 antibodies induced optimal activation.

As shown in FIGS. 21A and B, each of Bv8 and EG-VEGF induced a 5 to 8fold increase in 3H-thymidine incorporation into T cells. This dataindicates that Bv8 and EG-VEGF function as mitogens and potentialsurvival factors for CD4+ T cells.

Example 7 Induction of Cytokine Production EG-VEGF in CD4⁺ T Cells

The ability of EG-VEGF to induce cytokine production in CD4⁺ T cells wasstudied. CD4⁺ T cells were isolated and purified as described above forExample 6. T Cell proliferation assays were performed as described abovefor Example 6. Recombinant EG-VEGF was added to each well at aconcentration ranging from 2 to 200 nM. The wells were precoated for 2hours at 37° C. with 0.5 μg/ml anti-mouse CD3 antibody (Pharmingen,LaJolla, Calif.) and/or 1 μg/ml anti-mouse CD28 antibody (Pharmingen,LaJolla, Calif.) in carbonate buffer, pH 9.0. The antibodies crosslinkedthe respective receptors on the surface of the T cells, inducing basalactivation. Addition of anti-CD3 antibodies in combination withanti-CD28 antibodies induced optimal activation. Prior to the incubationstep, a 30 μl aliquot of the RPMI assay media was collected and replacedwith an equal volume of fresh assay media. During the 72 hour incubationstep, a 30 μl aliquot of the RPMI assay media was collected at 24 hourfor analysis of cytokine production and replaced with an equal volume offresh RPMI assay media. A second aliquot was collected immediately priorto the addition of ³H-thymidine. Analysis of the collected samples forIL-2 and IFN-γ was performed using the Luminex Multiplex Assay system(Luminex Corp., Austin, Tex.).

Cytokine production from CD4+ T cells was monitored followingstimulation with EG-VEGF. Within 24 hours of ligand addition, EG-VEGFinduced production of IL-2 (FIGS. 22A and B) and IFN-γ by the T cells(FIGS. 22C and D). The concentration of IFN-γ produced by the T cellsafter 72 hours of incubation with EG-VEGF exceeded the detection limitsof the assay. These results indicate that Bv8 and EG-VEGF are capable ofregulating the development of the CD4+ T cell response.

Example 8 Bv8 Promotes Recovery After 5-FU Myelosuppression

The ability of Bv8 to promote hematopoietic recovery aftermyelosuppression with 5-FU was studied. Recombinant adenoviruses wereproduced using the AdEasy vector system (Stratagene, LaJolla, Calif.).cDNA encoding an 81 amino acid isoform of mouse Bv8 (SEQ ID NO:6) orfull-length human EG-VEGF (SEQ ID NO:8) were cloned into the multiplecloning site of the pCMV shuttle vector. Recombination and subsequentamplification in 293 cells was performed as recommended by themanufacturer. The virus was purified from the supernatant and cellpellet using the AAV purification kit (Virapur, San Diego, Calif.).Virus titers were determined by standard plaque assays utilizingCMV-LacZ virus as a control. Additional controls included receptorselective mutants of VEGF.

Control nude mice were injected with recombinant adenoviruses via thetail vein. The virus dose for each animal was 10⁸ pfu in 100 microlitersof phosphate buffered saline. Blood cell counts were monitored for 12days following virus administration. Blood samples were obtained fromorbital sinus bleeds of the mice and analyzed using the Cell Dynautomated hematology analyzer (Abbott Diagnostics, Santa Clara, Calif.).Differential counts were performed manually in conjunction with theautomated analysis using light microscopy.

Test animals were injected with the recombinant adenovirus wasadministered to the mice 3 days prior to induction of myelosuppression.The virus dose for each animal was 10⁸ pfu in 100 microliters ofphosphate buffered saline. To induce myclosuppression in mice, a singledose of 125 mg/kg 5-fluorouracil (Adrucil, NDC 0013-1046-94) wasinjected in the peritoneum. Blood cell counts and differentials countswere measured 5, 10 and 14 days following administration of the 5-FU.Blood samples were obtained from orbital sinus bleeds of the mice andanalyzed using the Cell Dyn automated hematology analyzer (AbbottDiagnostics, Santa Clara, Calif.). Differential counts were performedmanually in conjunction with the automated analysis using lightmicroscopy. At 14 days following the administration 5-FU, animals weresacrificed and the spleens excised. The spleens were weighed and thenmechanically dissociated as described for Example 5. The spleencellularity was measured by counting cells in the single cell suspensionusing a Coulter counter (Beckman Coulter, Miama, Fla.). 2×10⁴ cells fromeach spleen were plated in triplicate in mouse complete Methocult (StemCell Technologies, Inc.) media and hematopoietic cell colony types werescored following 10-14 days in culture.

As shown in FIG. 23A, the white blood cell counts in the Bv8 treatmentgroup were significantly higher than other groups over the course of thestudy. Consistent with this data, granulocyte and monocyte numbers werealso higher (FIGS. 23B and C). Spleen cellularity was significantlygreater (at least 2.5-fold) in the Bv8 treated mice, than in controlgroups (FIG. 24). In addition, the Bv8 spleens contained a significantlyhigher number of progenitor cells than the non-virus or LacZ treatedmice (FIG. 24).

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. However,various modifications of the invention in addition to those shown anddescribed herein will be apparent to those skilled in the art from theforegoing description and fall within the scope of the appended claims.

1. A method of inducing proliferation of lymphoid lineage progenitorcells or progeny thereof, comprising contacting the lymphoid lineageprogenitor cells or progeny thereof with Bv8, or a combination of Bv8and EG-VEGF to induce proliferation of said cells, wherein said Bv8comprises at least 90% amino acid identity with SEQ ID NO:2 or SEQ IDNO:4 and wherein said EG-VEGF comprises at least 90% amino acid identitywith SEQ ID NO:8 or amino acids 20-105 of SEQ ID NO:8.
 2. The method ofclaim 1, wherein said progeny are lymphoid precursor cells orlymphocytes.
 3. The method of claim 2, wherein said progeny are T cells.4. The method of claim 3, wherein the T cells are CD4+ T cells.
 5. Themethod of claim 1, wherein the Bv8 comprises the amino acid sequence ofSEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6.
 6. The method of claim 1,wherein the Bv8 is a native human Bv8 polypeptide.
 7. The method ofclaim 1, wherein the Bv8 binds heparin.
 8. The method of claim 1,wherein the Bv8 comprises a fusion polypeptide, a chimeric polypeptide,or an immunoadhesin.
 9. The method of claim 1, wherein the EG-VEGF is anative human EG-VEGF polypeptide.
 10. The method of claim 1, wherein theEG-VEGF comprises SEQ ID NO:10 or amino acid residues 20-105 of SEQ IDNO:8.
 11. The method of claim 1, wherein the EG-VEGF comprises a fusionpolypeptide, a chimeric polypeptide, or an immunoadhesin.
 12. A methodof increasing the population of T lymphocytes in a subject, comprisingadministering Bv8, or a combination of Bv8 and EG-VEGF to a subjectfollowing treatment of the subject with an immunosuppressive agent,radiation, or chemotherapy, wherein said Bv8 comprises at least 90%amino acid identity with SEQ ID NO:2 or SEQ ID NO:4 and induces theproduction of T lymphocytes and wherein said EG-VEGF comprises at least90% amino acid identity with SEQ ID NO:8 or amino acids 20-105 of SEQ IDNO: 8 and induces the production of T lymphocytes.
 13. The method ofclaim 12, wherein the T lymphocytes are CD4+ T cells.
 14. The method ofclaim 12, wherein the chemotherapy comprises treatment with 5fluorouracil, vincristine, cisplatin, oxoplatin, methotrexate,3′-azido-3′-deoxythymidine, paclitaxel, doxetaxel, an anthracyclineantibiotic, or mixtures thereof.
 15. A method of increasing thepopulation of white blood cells in a subject, comprising administeringBv8 or a combination of Bv8 and EG-VEGF to a subject following treatmentof the subject with an immunosuppressive agent, radiation, orchemotherapy, wherein said Bv8 comprises at least 90% amino acididentity with SEQ ID NO:2 or SEQ ID NO:4 and induces the production ofwhite blood cells and wherein said EG-VEGF comprises at least 90% aminoacid identity with SEQ ID NO: 8 or amino acids 20-105 of SEQ ID NO:8 andinduces the production of white blood cells.
 16. The method of claim 15,wherein the white blood cells comprise neutrophils or B cells.
 17. Themethod of claim 15, wherein the chemotherapy comprises treatment with 5fluorouracil, vincristine, cisplatin, oxoplatin, methotrexate,3′-azido-3′-deoxythymidine, paclitaxel, doxetaxel, an anthracyclineantibiotic, or mixtures thereof